Mastering the canonical loop of serine protease inhibitors : enhancing potency by optimising the internal hydrogen bond network

Background Canonical serine protease inhibitors commonly bind to their targets through a rigid loop stabilised by an internal hydrogen bond network and disulfide bond(s). The smallest of these is sunflower trypsin inhibitor (SFTI-1), a potent and broad-range protease inhibitor. Recently, we re-engineered the contact β-sheet of SFTI-1 to produce a selective inhibitor of kallikrein-related peptidase 4 (KLK4), a protease associated with prostate cancer progression. However, modifications in the binding loop to achieve specificity may compromise structural rigidity and prevent re-engineered inhibitors from reaching optimal binding affinity. Methodology/Principal Findings In this study, the effect of amino acid substitutions on the internal hydrogen bonding network of SFTI were investigated using an in silico screen of inhibitor variants in complex with KLK4 or trypsin. Substitutions favouring internal hydrogen bond formation directly correlated with increased potency of inhibition in vitro. This produced a second generation inhibitor (SFTI-FCQR Asn14) which displayed both a 125-fold increased capacity to inhibit KLK4 (Ki = 0.0386±0.0060 nM) and enhanced selectivity over off-target serine proteases. Further, SFTI-FCQR Asn14 was stable in cell culture and bioavailable in mice when administered by intraperitoneal perfusion. Conclusion/Significance These findings highlight the importance of conserving structural rigidity of the binding loop in addition to optimising protease/inhibitor contacts when re-engineering canonical serine protease inhibitors.

ACE research vignette 017: The new venture mortality myth

This series of research vignettes is aimed at sharing current and interesting research findings from our team of internatinal Entrepreneurship researchers. In this vignette, Dr Jonathan Levie of the University of Strathclyde notes wide and persistent gaps between perceptions and measures of new business mortality, and discusses possible implications.

Re-cementing in revision total hip replacement

Removal of well-fixed cement at revision surgery risks bone loss, cortical perforation and fracture, is time-consuming, technically demanding and carries increased risks for the patient. The cement-in-cement technique avoids these problems and when used appropriately has given favourable results at our centre when used on both the femoral and acetabular sides of the articulation. A modified technique has also been used in selected cases of infection and peri-prosthetic fracture. This chapter highlights the results to date and the operative techniques employed. It is essential to recognise that this technique relies fundamentally on the presence of a well-fixed cement mantle, and it is imperative that the criteria laid out are adhered to in order to achieve success. If there is loosening or lysis on the femoral side extending distal to the lesser trochanter or around more than just the periphery of the acetabular cement mantle, then alternative revision techniques should be employed.

Novel methods and the maximum likelihood estimation technique for estimating traffic critical gap

Most unsignalised intersection capacity calculation procedures are based on gap acceptance models. Accuracy of critical gap estimation affects accuracy of capacity and delay estimation. Several methods have been published to estimate drivers’ sample mean critical gap, the Maximum Likelihood Estimation (MLE) technique regarded as the most accurate. This study assesses three novel methods; Average Central Gap (ACG) method, Strength Weighted Central Gap method (SWCG), and Mode Central Gap method (MCG), against MLE for their fidelity in rendering true sample mean critical gaps. A Monte Carlo event based simulation model was used to draw the maximum rejected gap and accepted gap for each of a sample of 300 drivers across 32 simulation runs. Simulation mean critical gap is varied between 3s and 8s, while offered gap rate is varied between 0.05veh/s and 0.55veh/s. This study affirms that MLE provides a close to perfect fit to simulation mean critical gaps across a broad range of conditions. The MCG method also provides an almost perfect fit and has superior computational simplicity and efficiency to the MLE. The SWCG method performs robustly under high flows; however, poorly under low to moderate flows. Further research is recommended using field traffic data, under a variety of minor stream and major stream flow conditions for a variety of minor stream movement types, to compare critical gap estimates using MLE against MCG. Should the MCG method prove as robust as MLE, serious consideration should be given to its adoption to estimate critical gap parameters in guidelines.