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.

Impact of the internal business environment on knowledge management within construction organisations

Purpose: Recent knowledge management (KM) literature suggests that KM activities are influenced by the elements of the internal business environment (BE) of organisations. This paper attempts to provide some unique insights into the contextual input of the KM process through empirically identifying the major factors (i.e. “forces”) within the internal BE of construction organisations operating in Hong Kong, and investigating their impact on the intensity of KM activities. Design/methodology/approach: A questionnaire survey was administered to a sample of construction contractors operating in Hong Kong to elicit opinions on the internal BE and intensity of KM activities as executed by targeted organisations. A total of 149 usable responses were received from 99 organisations representing about 38 percent of the research population. In parallel, to the survey, a total of 15 semi-structured interviews were undertaken to provide more insights into the phenomenon under investigation. Findings: Supported by the empirical and qualitative evidence, this study established that firstly, both organisational and technical environments have the capacity to either positively or negatively impact the intensity of KM activities, and both environments serve as stimuli in increasing each other's dynamism; secondly, certain types of KM activities are stronger “energy receivers” and easily to be “powered up” by manipulating factors representing these two environments. Then, through interactions between KM activities, the intensity of the whole strategic KM cycle will be increased thus helping to strengthen organisational competitive advantage.

Permeability of high-performance concrete incorporating presoaked lightweight aggregates for internal curing

This paper presents an experimental study on the effect of presoaked lightweight aggregates (LWAs) for internal curing on water permeability, water absorption and resistance of concrete to chloride-ion penetration in comparison with those of a control concrete and a concrete with shrinkage reducing admixture (SRA) of similar water/cement ratios (w/c). In general, the concretes with LWA particles had initial water absorption, sorptivity and water permeability similar to or lower than those of the control concrete and the concrete with SRA. The charges passed, chloride migration coefficient and chloride diffusion coefficient of such concretes were in the same order as those of the control concrete and the concrete with SRA. However, the incorporation of the LWAs for internal curing reduced unit weight, compressive strength and elastic modulus of the concrete. Comparing the LWAs of different sizes for internal curing, finer particles were more efficient in reducing the shrinkage and generally resulted in less reduction in the unit weight, compressive strength, and elastic modulus. However, the increase in the more porous crushed LW particles in concrete seems to increase the penetration of chloride ions in the concrete. The concrete with SRA had initial water absorption, sorptivity, water permeability and resistance to chloride ion penetration comparable with those of the control concrete. The use of SRA in concrete does not affect the elastic modulus of the concrete, except for a minor influence on the compressive strength of the concrete.

A retrospective cohort investigation of active range of motion within one week of open reduction and internal fixation of distal radius fractures

Distal radius fractures stabilized by open reduction internal fixation (ORIF) have become increasingly common. There is currently no consensus on the optimal time to commence range of motion (ROM) exercises post-ORIF. A retrospective cohort review was conducted over a five-year period to compare wrist and forearm range of motion outcomes and number of therapy sessions between patients who commenced active ROM exercises within the first seven days and from day eight onward following ORIF of distal radius fractures. One hundred and twenty-one patient cases were identified. Clinical data, active ROM at initial and discharge therapy assessments, fracture type, surgical approaches, and number of therapy sessions attended were recorded. One hundred and seven (88.4%) cases had complete datasets. The early active ROM group (n = 37) commenced ROM a mean (SD) of 4.27 (1.8) days post-ORIF. The comparator group (n = 70) commenced ROM exercises 24.3 (13.6) days post-ORIF. No significant differences were identified between groups in ROM at initial or discharge assessments, or therapy sessions attended. The results from this study indicate that patients who commenced active ROM exercises an average of 24 days after surgery achieved comparable ROM outcomes with similar number of therapy sessions to those who commenced ROM exercises within the first week.

β-Actin—an unsuitable internal control for RT-PCR

Despite reports confirming cell-cycle dependent gene expression and a number of studies describing specific circumstances in which β-actin is also regulated, the mRNA for β-actin remains a widely used housekeeping gene internal control. Utilizing differential reverse transcriptase-polymerase chain reaction (RT-PCR), we report here the dose-dependent inhibition of β-actin by matrigel. This was detected by comparison to the very moderate inhibition of the target gene, membrane type-1 matrix metalloproteinase (MT1-MMP), with results independently confirmed by similar findings on MT1-MMP expression using competitive RT-PCR. Furthermore, RT-PCR of the housekeeping gene 18 Svedberg Units (S) rRNA demonstrated excellent consistency, reproducibility and non-regulation by a matrigel treatment. We conclude that β-actin is highly regulated by matrigel and therefore unsuitable as an internal control in this treatment. Hence, these findings suggest that researchers have a responsibility to ensure that the housekeeping gene of choice is not regulated in their specific application, as such regulation may dramatically affect the accuracy of their results. This study reinforces the necessity for minimally regulated housekeeping genes such as 18S rRNA, and the superiority of competitive templates as internal controls for quantitative applications of RT-PCR.

Predicting the fatigue life of internal fracture fixation plates

Failures of fracture fixation plates, often related to fatigue fractures of the implants, have been reported (Banovetz et al, 1996). While metallurgical defects can usually be excluded, many of these fractures can be explained with the biomechanical situation. This study investigated the biomechanics of two clinical cases, both of which used a 14-hole locking compression plate. In the first case, a titanium plate was used in a rigid configuration with 12 screws resulting in plate breakage after 7 weeks (Sommer et al, 2003). In the second case, a stainless steel plate, which endured the entire healing process, was used in a flexible application with only 6 screws.