Using the forces of hydrodynamic countercurrent chromatography for the study of bacteriophages.

Bacteriophages (phages) are viruses that target bacteria, with the ability to lyse and kill host bacterial cells. Due to this, they have been of some interest as a therapeutic since their discovery in the early 1900s, but with the recent increase in antibiotic resistance, phages have seen a resurgence in attention. Current methods of isolation and purification of phages can be long and tedious, with caesium chloride concentration gradients the gold standard for purifying a phage fraction. Isolation of novel phages requires centrifugation and ultrafiltration of mixed samples, such as water sources, effluent or faecal samples etc, to prepare phage filtrates for further testing. We propose countercurrent chromatography as a novel and alternative approach to use when studying phages, as a scalable and high-yield method for obtaining phage fractions. However, the full extent of the usefulness and resolution of separation with this technique has not been researched; it requires optimization and ample testing before this can be revealed. Here we present an initial study to determine survivability of two phages, T4 and ϕX174, using only water as a mobile phase in a Spectrum Series 20 HPCCC. Both phages were found to remain active once eluted from the column. Phages do not fully elute from the column and sodium hydroxide is necessary to flush the column between runs to deactivate remaining phages.

Fatigue and damage of porcine pars interarticularis during asymmetric loading.

If the articular facets of the vertebra grow in an asymmetric manner, the developed bone geometry causes an asymmetry of loading. When the loading environment is altered by way of increased activity, the likelihood of acquiring a stress fracture may be increased. The combination of geometric asymmetry and increased activity is hypothesised to be the precursor to the stress fracture under investigation in this study, spondylolysis. This vertebral defect is an acquired fracture with 7% prevalence in the paediatric population. This value increases to 21% among athletes who participate in hyperextension sports. Tests were carried out on porcine lumbar vertebrae, on which the effect of facet angle asymmetry was simulated by offsetting the load laterally by 7mm from the mid-point. Strain in the vertebral laminae was recorded using six 3-element stacked rosette strain gauges placed bilaterally. Specimens were loaded cyclically at a rate of 2Hz. Fatigue cycles; strain, creep, secant modulus and hysteresis were measured. The principal conclusions of this paper are that differences in facet angle lead to an asymmetry of loading in the facet joints; this in turn leads to an initial increase in strain on the side with the more coronally orientated facet. The strain amplitude, which is the driving force for crack propagation, is greater on this side at all times up to fracture, the significance of this can be observed in the increased steady state creep rate (p = 0.036) and the increase in yielding and toughening mechanisms taking place, quantified by the force-displacement hysteresis (p = 0.026).

Strain distribution in the porcine lumbar laminae under asymmetric loading.

If the articular facets of the vertebra grow in an asymmetric manner, the developed geometry causes an asymmetry of loading. When the loading environment is altered by way of increased activity, the likelihood of acquiring a stress fracture may be increased. The combination of geometric asymmetry and increased activity is hypothesised to be the precursor to the stress fracture under investigation in this study, spondylolysis. This vertebral defect is an acquired fracture with 7% prevalence in the paediatric population. This value increases to 21% among athletes who participate in hyperextension sports. Tests were carried out on porcine lumbar vertebrae, on which the effect of facet angle asymmetry was simulated by offsetting the load laterally by 7 mm from the mid-point. The aim of the study is to investigate whether an increase in the coronal orientation of one facet leads to an increase in strain in the corresponding vertebral lamina. Strain in the laminae was recorded using six 3-element stacked rosette strain gauges placed bilaterally. Results show that a significant linear predictive relationship exists between load offset and average strain level in the vertebral laminae with p values of 0.006 and 0.045 for principal strains ε1 and ε2 on the right-hand side, and p-values of 0.009 and 0.001 for principal strains ε1 and ε2 on the left-hand side ( R2 all >0.9). This study concludes that facet angle asymmetry does lead to a difference in strain in the vertebral laminae. Change in principal strain as a result of facet asymmetry has a linear relationship and an asymmetry threshold exists beyond which compressive strain on the more coronally oriented facet can be increased by up to 15%.

1H-Pyrazolo[3,4-g]hexahydro-isoquinolines as selective glucocorticoid receptor antagonists with high functional activity.

Addition of the 4-fluorophenylpyrazole group to the previously described 2-azadecalin glucocorticoid receptor (GR) antagonist 1 resulted in significantly enhanced functional activity. SAR of the bridgehead substituent indicated that whereas groups as small as methyl afforded high GR binding, GR functional activity was enhanced by larger groups such as benzyl, substituted ethers, and aminoalkyl derivatives. GR antagonists with binding and functional activity comparable to mifepristone were discovered (e.g., 52: GR binding K(i) 0.7 nM; GR reporter gene functional K(i) 0.6 nM) and found to be highly selective over other steroid receptors. Analogues 43 and 45 had >50% oral bioavailability in the dog.

Mapping the kinase domain of Janus Kinase 3.

The utilization and impact of parallel synthesis on lead exploration around initial hit oxindole (1) are described. The emergent SAR, analogue design and functional impact will also be detailed.