The efficacy of a video-based marker-less tracking system for gait analysis

Most clinical gait analyses are conducted using motion capture systems which track retro-reflective markers that are placed on key landmarks of the participants. An alternative to a three-dimensional (3D) motion capture, marker-based, optical camera system may be a marker-less video-based tracking system. The aim of our study was to investigate the efficacy of the use of a marker-less tracking system in the calculation of 3D joint angles for possible use in clinical gait analysis. Ten participants walked and jogged on a treadmill and their kinematic data were captured with a marker and marker-less tracking system simultaneously. The hip, knee and ankle angles in the frontal, sagittal and transverse planes were computed. Root Mean Square differences (RMS_diff) between corresponding angles for each participant’s support phase were calculated and averaged to derive the mean within-subject RMS_diff. These within-subject means were averaged to obtain the mean between-subject RMS_diff for the relevant joint angles in the two gait conditions (walking and jogging). The RMS_diff between the two tracking systems was less than 1° for all rotations of the three joint angles of the hip and knee. However, there were slightly larger differences in the ankle joint angles. The results of this study suggest a potential application in gait analysis in clinical settings where observations of anatomical motions may provide meaningful feedback.     

Activation loop targeting strategy for design of receptor-interacting protein kinase 2 (RIPK2) inhibitors

Development of selective kinase inhibitors remains a challenge due to considerable amino acid sequence similarity among family members particularly in the ATP binding site. Targeting the activation loop might offer improved inhibitor selectivity since this region of kinases is less conserved. However, the strategy presents difficulties due to activation loop flexibility. Herein, we report the design of receptor-interacting protein kinase 2 (RIPK2) inhibitors based on pan-kinase inhibitor regorafenib that aim to engage basic activation loop residues Lys169 or Arg171. We report development of CSR35 that displayed >10-fold selective inhibition of RIPK2 versus VEGFR2, the target of regorafenib. A co-crystal structure of CSR35 with RIPK2 revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in the inhibitor and the side-chain of Lys169. Our data provides principle feasibility of developing activation loop targeting type II inhibitors as a complementary strategy for achieving improved selectivity.     

Combined NMR and EPR spectroscopy to determine structures of viral fusion domains in membranes

Methods are described to determine the structures of viral membrane fusion domains in detergent micelles by NMR and in lipid bilayers by site-directed spin labeling and EPR spectroscopy. Since in favorable cases, the lower-resolution spin label data obtained in lipid bilayers fully support the higher-resolution structures obtained by solution NMR, it is possible to graft the NMR structural coordinates into membranes using the EPR-derived distance restraints to the lipid bilayer. Electron paramagnetic dynamics and distance measurements in bilayers support conclusions drawn from NMR in detergent micelles. When these methods are applied to a structure determination of the influenza virus fusion domain and four point mutations with different functional phenotypes, it is evident that a fixed-angle boomerang structure with a glycine edge on the outside of the N-terminal arm is both necessary and sufficient to support membrane fusion. The human immunodeficiency virus fusion domain forms a straight helix with a flexible C-terminus. While EPR data for this fusion domain are not yet available, it is tentatively speculated that, because of its higher hydrophobicity, a critically tilted insertion may occur even in the absence of a kinked boomerang structure in this case.     

Can the invasive European rabbit (Oryctolagus cuniculus) assume the soil engineering role of locally-extinct natives?

Habitat modifying species can play crucial roles in ecosystem function. Invasive engineers may assume these roles where native engineers have been lost from the system. We compared the dynamics of the foraging pits of an invasive engineer, the European rabbit (Oryctolagus cuniculus) with two native mammals, the greater bilby (Macrotis lagotis) and the burrowing bettong (Bettongia lesueur). Foraging pits are small surface depressions created by animals when they forage for seeds, bulbs, roots, invertebrates and fungi. We measured foraging pit density and turnover, and density and richness of plant seedlings in pits and adjacent surfaces across three landforms representing a gradient in resource availability inside (bilbies and bettongs), and outside (rabbits only) a reserve in an arid Australian shrubland over 2 years. Pits of the native engineers contained 80% more seedlings (11.2 plants m⁻²) than rabbit pits (6.22 plants m⁻²). Further, rabbit pits supported 3.6-times fewer seedlings than equivalent non-pit surfaces outside the exclosure. Only one plant species was restricted entirely to pits. The reserve had more foraging pits and greater turnover than outside, but contrary to prediction, pit effects on seedling density were no greater in the more resource-limited dunes. There were some strong temporal and landscape effects on pit density and species composition, but generally trends were similar inside and outside the reserve. Overall, despite their functional similarities, invasive rabbits created fewer pits that were less favourable patches for seedlings than those of native engineers. Our work suggests that a suite of ecosystem processes associated with fertile patch creation has potentially been lost with the extirpation of bilbies and bettongs.     

The spatial arrangement of chromosomes during prometaphase facilitates spindle assembly

Error-free chromosome segregation requires stable attachment of sister kinetochores to the opposite spindle poles (amphitelic attachment). Exactly how amphitelic attachments are achieved during spindle assembly remains elusive. We employed photoactivatable GFP and high-resolution live-cell confocal microscopy to visualize complete 3D movements of individual kinetochores throughout mitosis in nontransformed human cells. Combined with electron microscopy, molecular perturbations, and immunofluorescence analyses, this approach reveals unexpected details of chromosome behavior. Our data demonstrate that unstable lateral interactions between kinetochores and microtubules dominate during early prometaphase. These transient interactions lead to the reproducible arrangement of chromosomes in an equatorial ring on the surface of the nascent spindle. A computational model predicts that this toroidal distribution of chromosomes exposes kinetochores to a high density of microtubules which facilitates subsequent formation of amphitelic attachments. Thus, spindle formation involves a previously overlooked stage of chromosome prepositioning which promotes formation of amphitelic attachments.     

Degradation of some phenols and hydroxybenzoates by the imperfect ascomycetous yeastsCandida parapsilosis andArxula adeninivorans: evidence for an operative gentisate pathway

The imperfect ascomycetous yeastsCandida parapsilosis andArxula adeninivorans degraded 3-hydroxybenzoic acid via gentisate which was the cleavage substrate. 4-Hydroxybenzoic acid was metabolized via protocatechuate. No cleavage enzyme for the latter was detected. In stead of this NADH- and NADPH-dependent monooxygenases were present. In cells grown at the expense of hydroquinone and 4-hydroxygenzoic acid, enzymes of the hydroxyhydroquinone variant of the 3-oxoadipate pathway were demonstrated, which also took part in the degradation of 2,4-dihydroxybenzoic acid byC. parapsilosis.Abbreviations HHQ Hydroxyhydroquinone (1,2,4-trihydroxybenzene) - GSH reduced Glutathione     

PCR fingerprinting for epidemiological studies of Staphylococcus aureus

Staphylococcus aureus isolates (n = 126), collected during two different periods from patients hospitalised in pediatric wards, were analysed using polymerase chain reaction (PCR) mediated genotyping. These isolates were compared with 29 isolates from individuals attending the out-patient clinic of the same hospital and 13 isolates from pediatric hospital personnel. Within a group of 99 isolates gathered from 48 individuals during surveillance period I, 22 distinct genotypes were identified by application of two PCR assays. Among the 58 isolates collected in surveillance period II from pediatric and out-clinic patients, 25 genotypes were detected by a single PCR assay only. Based on these results it was demonstrated that patients can be colonised with multiple strains that may persist in a certain anatomical location for prolonged periods of time. It is shown that persistence of a S. aureus strain in a pediatric ward can be deduced from the PCR genotyping studies. As such PCR can be used for longitudinal monitoring of bacterial infections in hospital departments, analysis of patient-to-patient and personnel-to-patient transmission and for detection of genetic variation in general in S. aureus. Also, isolate-specific DNA probes can be generated for S. aureus by PCR genotyping. The probes can be used for the recognition of re-emerging S. aureus epidemics.