Construction and Immunogenicity Evaluation of Recombinant Influenza A Viruses Containing Chimeric Hemagglutinin Genes Derived from Genetically Divergent Influenza A H1N1 Subtype Viruses

Background and Objectives

Influenza A viruses cause highly contagious diseases in a variety of hosts, including humans and pigs. To develop a vaccine that can be broadly effective against genetically divergent strains of the virus, in this study we employed molecular breeding (DNA shuffling) technology to create a panel of chimeric HA genes.

Methods and Results

Each chimeric HA gene contained genetic elements from parental swine influenza A viruses that had a history of zoonotic transmission, and also from a 2009 pandemic virus. Each parental virus represents a major phylogenetic clade of influenza A H1N1 viruses. Nine shuffled HA constructs were initially screened for immunogenicity in mice by DNA immunization, and one chimeric HA (HA-129) was expressed on both a A/Puerto Rico/8/34 backbone with mutations associated with a live, attenuated phenotype (PR8_LAIV-129) and a A/swine/Texas/4199-2/98 backbone (TX98-129). When delivered to mice, the PR8_LAIV-129 induced antibodies against all four parental viruses, which was similar to the breadth of immunity observed when HA-129 was delivered as a DNA vaccine. This chimeric HA was then tested as a candidate vaccine in a nursery pig model, using inactivated TX98-129 virus as the backbone. The results demonstrate that pigs immunized with HA-129 developed antibodies against all four parental viruses, as well as additional primary swine H1N1 influenza virus field isolates.

Conclusion

This study established a platform for creating novel genes of influenza viruses using a molecular breeding approach, which will have important applications toward future development of broadly protective influenza virus vaccines.     

Comparison of NK-1 Receptor Antagonist (Maropitant) to Morphine as a Pre-Anaesthetic Agent for Canine Ovariohysterectomy

Objective

To compare the NK-1 receptor antagonist maropitant to morphine during and after surgery in dogs undergoing ovariohysterectomy (OHE).

Methods

30 healthy female dogs were randomly divided to receive either a pre-anaesthetic dose of morphine (0.5 mg/kg SQ) or maropitant (1 mg/kg, SQ) prior to OHE. Anaesthesia was induced with propofol and maintained with isoflurane. Expired isoflurane concentration, heart rate (HR), systolic arterial pressure (SAP) and respiratory rate were measured. Post-operative pain scores and appetite were evaluated during the recovery period. Rescue analgesia (morphine 0.1 mg/kg IV) was administered as needed post-operatively based on blinded pain score assessments.

Results

Although clinically comparable; during surgical stimulation, the maropitant group had lower HR (108±18 vs 115±24 bpm; p = 0.04), lower SAP (114±23 vs 125±23 mmHg; p = 0.003) and required slightly lower percent of isoflurane anaesthetic (1.35±0.2 vs 1.51±0.4%; p = 0.005), when compared to the morphine group. In the recovery period, the maropitant group had lower pain scores at extubation (1.7±0.7 vs 3.4±2.3; p = 0.0001) and were more likely to eat within 3 hours after extubation (64.7 vs 15.3%). However, post-operative rescue analgesia requirements were similar between groups. All other measured parameters were similar between groups. The overall difference observed between groups was small and all monitored and measured parameters were within the expected range for anesthetized dogs.

Clinical Significance

No major differences in cardiorespiratory parameters or anaesthetic requirements were observed between maropitant and morphine when used as a pre-anesthetic agent for OHE. Further studies are necessary to fully elucidate the benefits of maropitant as a pre-anaesthetic agent for canine OHE.     

Inflammatory Bowel Disease and Risk of Adverse Pregnancy Outcomes

Background and Objectives

Existing data on pregnancy complications in inflammatory bowel disease (IBD) are inconsistent. To address these inconsistencies, we investigated potential associations between IBD, IBD-related medication use during pregnancy, and pregnancy loss, pre-eclampsia, preterm delivery, Apgar score, and congenital abnormalities.

Methods

We conducted a cohort study in >85,000 Danish National Birth Cohort women who were pregnant in the period 1996-2002 and had information on IBD, IBD-related medication use (systemic or local corticosteroids, 5-aminosalicylates), pregnancy outcomes and potential confounders. We evaluated associations between IBD and adverse pregnancy/birth outcomes using Cox regression and log-linear binomial regression.

Results

IBD was strongly and significantly associated with severe pre-eclampsia, preterm premature rupture of membranes and medically indicated preterm delivery in women using systemic corticosteroids during pregnancy (hazard ratios [HRs] >7). IBD was also associated with premature preterm rupture of membranes in women using local corticosteroid medications (HR 3.30, 95% confidence interval [CI] 1.33-8.20) and with medically indicated preterm delivery (HR 1.91, 95% CI 0.99-3.68) in non-medicated women. Furthermore, IBD was associated with low 5-minute Apgar score in term infants (risk ratio [RR] 2.19, 95% CI 1.03-4.66). Finally, Crohn’s disease (but not ulcerative colitis) was associated with major congenital abnormalities in the offspring (RR 1.85, 95% CI 1.06-3.21). No child with a congenital abnormality born to a woman with IBD was exposed to systemic corticosteroids in utero.

Conclusion

Women with IBD are at increased risk of severe pre-eclampsia, medically indicated preterm delivery, preterm premature rupture of membranes, and delivering infants with low Apgar score and major congenital malformations. These associations are only partly explained by severe disease as reflected by systemic corticosteroid use.     

The Role of Compassion in Altruistic Helping and Punishment Behavior

Compassion, the emotional response of caring for another who is suffering and that results in motivation to relieve suffering, is thought to be an emotional antecedent to altruistic behavior. However, it remains unclear whether compassion enhances altruistic behavior in a uniform way or is specific to sub-types of behavior such as altruistic helping of a victim or altruistic punishment of a transgressor. We investigated the relationship between compassion and subtypes of altruistic behavior using third-party paradigms where participants 1) witnessed an unfair economic exchange between a transgressor and a victim, and 2) had the opportunity to either spend personal funds to either economically a) help the victim or b) punish the transgressor. In Study 1, we examined whether individual differences in self-reported empathic concern (the emotional component of compassion) was associated with greater altruistic helping or punishment behavior in two independent samples. For participants who witnessed an unfair transaction, trait empathic concern was associated with greater helping of a victim and had no relationship to punishment. However, in those who decided to punish the transgressor, participants who reported greater empathic concern decided to punish less. In Study 2, we directly enhanced compassion using short-term online compassion meditation training to examine whether altruistic helping and punishment were increased after two weeks of training. Compared to an active reappraisal training control group, the compassion training group gave more to help the victim and did not differ in punishment of the transgressor. Together, these two studies suggest that compassion is related to greater altruistic helping of victims and is not associated with or may mitigate altruistic punishment of transgressors.     

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.     

Molecular evidence for hybridization in Colias (Lepidoptera: Pieridae): are Colias hybrids really hybrids?

Gene flow and hybridization among species dramatically affect our understanding of the species as a biological unit, species relationships, and species adaptations. In North American Colias eurytheme and Colias eriphyle, there has been historical debate over the extent of hybridization occurring and the identity of phenotypically intermediate individuals as genetic hybrids. This study assesses the population structure of these two species to measure the extent of hybridization and the genetic identity of phenotypic intermediates as hybrids. Amplified fragment length polymorphism (AFLP) marker analysis was performed on 378 specimens collected from northern California and Nevada. Population structure was inferred using a Bayesian/Markov chain Monte Carlo method, which probabilistically assigns individuals to genetic clusters. Three genetic clusters provided the best fit for the data. C. eurytheme individuals were primarily assigned to two closely related clusters, and C. eriphyle individuals were mostly assigned to a third, more distantly related cluster. There appeared to be significant hybridization between the two species. Individuals of intermediate phenotype (putative hybrids) were found to be genetically indistinguishable from C. eriphyle, indicating that previous work based on the assumption that these intermediate forms are hybrids may warrant reconsideration.     

Effects of the Epichloë fungal endophyte symbiosis with Schedonorus pratensis on host grass invasiveness

Initial studies of grass–endophyte mutualisms using Schedonorus arundinaceus cultivar Kentucky‐31 infected with the vertically transmitted endophyte Epichloë coenophiala found strong, positive endophyte effects on host‐grass invasion success. However, more recent work using different cultivars of S. arundinaceus has cast doubt on the ubiquity of this effect, at least as it pertains to S. arundinaceus–E. coenophiala. We investigated the generality of previous work on vertically transmitted Epichloë‐associated grass invasiveness by studying a pair of very closely related species: S. pratensis and E. uncinata. Seven cultivars of S. pratensis and two cultivars of S. arundinaceus that were developed with high‐ or low‐endophyte infection rate were broadcast seeded into 2 × 2‐m plots in a tilled, old‐field grassland community in a completely randomized block design. Schedonorus abundance, endophyte infection rate, and co‐occurring vegetation were sampled 3, 4, 5, and 6 years after establishment, and the aboveground invertebrate community was sampled in S. pratensis plots 3 and 4 years after establishment. Endophyte infection did not enable the host grass to achieve high abundance in the plant community. Contrary to expectations, high‐endophyte S. pratensis increased plant richness relative to low‐endophyte cultivars. However, as expected, high‐endophyte S. pratensis marginally decreased invertebrate taxon richness. Endophyte effects on vegetation and invertebrate community composition were inconsistent among cultivars and were weaker than temporal effects. The effect of the grass–Epichloë symbiosis on diversity is not generalizable, but rather specific to species, cultivar, infection, and potentially site. Examining grass–endophyte systems using multiple cultivars and species replicated among sites will be important to determine the range of conditions in which endophyte associations benefit host grass performance and have subsequent effects on co‐occurring biotic communities.     

Recombinase‐mediated cassette exchange (RMCE) for monoclonal antibody expression in the commercially relevant CHOK1SV cell line

To meet product quality and cost parameters for therapeutic monoclonal antibody (mAb) production, cell lines are required to have excellent growth, stability, and productivity characteristics. In particular, cell line generation stability is critical to the success of a program, especially where high cell line generation numbers are required for large in‐market supply. However, a typical process for developing such cell lines is laborious, lengthy, and costly. In this study, we applied a FLP/FRT recombinase‐mediated cassette exchange (RMCE) system to build a site‐specific integration (SSI) system for mAb expression in the commercially relevant CHOK1SV cell line. Using a vector with a FRT‐flanked mAb expression cassette, we generated a clonal cell line with good productivity, long‐term production stability, and low mAb gene‐copy number indicating the vector was located in a ‘hot‐spot.’ A SSI host cell line was made by removing the mAb genes from the ‘hot‐spot’ by RMCE, creating a ‘landing pad’ containing two recombination cassettes that allow targeting of one or two copies of recombinant genes. Cell lines made from this host exhibited excellent growth and productivity profiles, and stability for at least 100 generations in the absence of selection agents. Importantly, while clones containing two copies had higher productivity than single copy clones, both were stable over many generations. Taken together, this study suggests the use of FLP‐based RMCE to develop SSI host cells for mAb production in CHOK1SV offers significant savings in both resources and overall cell line development time, leading to a shortened ‘time‐to‐clinic’ for therapeutic mAbs. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1645–1656, 2015     

Evidence of Tree Species’ Range Shifts in a Complex Landscape

Climate change is expected to change the distribution of species. For long-lived, sessile species such as trees, tracking the warming climate depends on seedling colonization of newly favorable areas. We compare the distribution of seedlings and mature trees for all but the rarest tree species in California, Oregon and Washington, United States of America, a large, environmentally diverse region. Across 46 species, the mean annual temperature of the range of seedlings was 0.120°C colder than that of the range of trees (95% confidence interval from 0.096 to 0.144°C). The extremes of the seedling distributions also shifted towards colder temperature than those of mature trees, but the change was less pronounced. Although the mean elevation and mean latitude of the range of seedlings was higher than and north of those of the range of mature trees, elevational and latitudinal shifts run in opposite directions for the majority of the species, reflecting the lack of a direct biological relationship between species’ distributions and those variables. The broad scale, environmental diversity and variety of disturbance regimes and land uses of the study area, the large number and exhaustive sampling of tree species, and the direct causal relationship between the temperature response and a warming climate, provide strong evidence to attribute the observed shifts to climate change.     

Under What Circumstances Do Wood Products from Native Forests Benefit Climate Change Mitigation?

Climate change mitigation benefits from the land sector are not being fully realised because of uncertainty and controversy about the role of native forest management. The dominant policy view, as stated in the IPCC’s Fifth Assessment Report, is that sustainable forest harvesting yielding wood products, generates the largest mitigation benefit. We demonstrate that changing native forest management from commercial harvesting to conservation can make an important contribution to mitigation. Conservation of native forests results in an immediate and substantial reduction in net emissions relative to a reference case of commercial harvesting. We calibrated models to simulate scenarios of native forest management for two Australian case studies: mixed-eucalypt in New South Wales and Mountain Ash in Victoria. Carbon stocks in the harvested forest included forest biomass, wood and paper products, waste in landfill, and bioenergy that substituted for fossil fuel energy. The conservation forest included forest biomass, and subtracted stocks for the foregone products that were substituted by non-wood products or plantation products. Total carbon stocks were lower in harvested forest than in conservation forest in both case studies over the 100-year simulation period. We tested a range of potential parameter values reported in the literature: none could increase the combined carbon stock in products, slash, landfill and substitution sufficiently to exceed the increase in carbon stock due to changing management of native forest to conservation. The key parameters determining carbon stock change under different forest management scenarios are those affecting accumulation of carbon in forest biomass, rather than parameters affecting transfers among wood products. This analysis helps prioritise mitigation activities to focus on maximising forest biomass. International forest-related policies, including negotiations under the UNFCCC, have failed to recognize fully the mitigation value of native forest conservation. Our analyses provide evidence for decision-making about the circumstances under which forest management provides mitigation benefits.