Improving the pharmacokinetic properties of biologics by fusion to an anti-HSA shark VNAR domain

Advances in recombinant antibody technology and protein engineering have provided the opportunity to reduce antibodies to their smallest binding domain components and have concomitantly driven the requirement for devising strategies to increase serum half-life to optimise drug exposure, thereby increasing therapeutic efficacy. In this study, we adopted an immunization route to raise picomolar affinity shark immunoglobulin new antigen receptors (IgNARs) to target human serum albumin (HSA). From our model shark species, Squalus acanthias, a phage display library encompassing the variable binding domain of IgNAR (VNAR) was constructed, screened against target, and positive clones were characterized for affinity and specificity. N-terminal and C-terminal molecular fusions of our lead hit in complex with a naïve VNAR domain were expressed, purified and exhibited the retention of high affinity binding to HSA, but also cross-selectivity to mouse, rat and monkey serum albumin both in vitro and in vivo. Furthermore, the naïve VNAR had enhanced pharmacokinetic (PK) characteristics in both N- and C-terminal orientations and when tested as a three domain construct with naïve VNAR flanking the HSA binding domain at both the N and C termini. Molecules derived from this platform technology also demonstrated the potential for clinical utility by being available via the subcutaneous route of delivery. This study thus demonstrates the first in vivo functional efficacy of a VNAR binding domain with the ability to enhance PK properties and support delivery of multifunctional therapies.     

The role of oxygen availability in the embryonation of Heterakis gallinarum eggs.

The importance of oxygen availability in the embryonation of the infective egg stages of the gastrointestinal nematode parasite Heterakis gallinarum was studied in the laboratory. Unembryonated H. gallinarum eggs were kept under either aerobic conditions by gassing with oxygen, or anaerobic conditions by gassing with the inert gas nitrogen, under a range of constant temperatures. Oxygenated eggs embryonated at a rate influenced by temperature. Conversely, eggs treated with nitrogen showed no embryonation although when these eggs were transferred from nitrogen to oxygen gas after 60 days of treatment, embryonation occurred. This demonstrated that oxygen is an essential requirement for H. gallinarum egg development, although undeveloped eggs remain viable, even after 60 days in low oxygen conditions. The effects of climate on the biology of free-living stages studied under constant laboratory conditions cannot be applied directly to the field where climatic factors exhibit daily cycles. The effect of fluctuating temperature on development was investigated by including an additional temperature group in which H. gallinarum eggs were kept under daily temperature cycles between 12 and 22°C. Cycles caused eggs to develop significantly earlier than those in the constant mean cycle temperature, 17°C, but significantly slower than those in constant 22°C suggesting that daily temperature cycles had an accelerating effect on H. gallinarum egg embryonation but did not accelerate to the higher temperature. These results suggest that daily fluctuations in temperature influence development of the free-living stages and so development cannot be accurately predicted on the basis of constant temperature culture.     

Focal Plant Observations as a Standardised Method for Pollinator Monitoring: Opportunities and Limitations for Mass Participation Citizen Science

BackgroundRecently there has been increasing focus on monitoring pollinating insects, due to concerns about their declines, and interest in the role of volunteers in monitoring pollinators, particularly bumblebees, via citizen science.

Methodology / Principal Findings

The Big Bumblebee Discovery was a one-year citizen science project run by a partnership of EDF Energy, the British Science Association and the Centre for Ecology & Hydrology which sought to assess the influence of the landscape at multiple scales on the diversity and abundance of bumblebees. Timed counts of bumblebees (Bombus spp.; identified to six colour groups) visiting focal plants of lavender (Lavendula spp.) were carried out by about 13 000 primary school children (7–11 years old) from over 4000 schools across the UK. 3948 reports were received totalling 26 868 bumblebees. We found that while the wider landscape type had no significant effect on reported bumblebee abundance, the local proximity to flowers had a significant effect (fewer bumblebees where other flowers were reported to be >5m away from the focal plant). However, the rate of mis-identifcation, revealed by photographs uploaded by participants and a photo-based quiz, was high.

Conclusions / Significance

Our citizen science results support recent research on the importance of local flocal resources on pollinator abundance. Timed counts of insects visiting a lure plant is potentially an effective approach for standardised pollinator monitoring, engaging a large number of participants with a simple protocol. However, the relatively high rate of mis-identifications (compared to reports from previous pollinator citizen science projects) highlights the importance of investing in resources to train volunteers. Also, to be a scientifically valid method for enquiry, citizen science data needs to be sufficiently high quality, so receiving supporting evidence (such as photographs) would allow this to be tested and for records to be verified.     

Dominant lethal studies in male mice after exposure to 2.45 GHz microwave radiation

Adult male mice had the lower halves of their bodies exposed in a waveguide system to 2.45 GHz microwave radiation for 30 min. The half body dose-rate of 43 W kg-1 had been shown in a previous study [7] to deplete severely the heat-sensitive stages of sperm production. The males were mated at intervals to adult hybrid females over the following 8-10 weeks. There was no significant reduction in post-implantation survival, suggesting that the microwave exposure did not have a mutagenic effect on the male germ cells. However, pregnancy rate was significantly reduced in weeks 3, 4, 5 and 6; reaching a minimum of about 10% of the control value in weeks 4 and 5. The occurrence of low values in weeks 4 and 5 correlated well with the expected reductions in sperm count due to the pattern of depletion of the spermatogenic epithelium of the testes. Thus it was concluded that the reduced pregnancy rate resulted from reduced male fertility. Pre-implantation survival can also be affected by reduced sperm count [8] and was significantly reduced in this study but it correlated less well with the anticipated heat response. A further study is in progress looking at the contribution of sperm count and sperm abnormality to the results.