Urodynamic parameters and plasma LH/FSH in spayed Beagle bitches before and 8 weeks after GnRH depot analogue treatment

The pathophysiology of urinary incontinence due to spaying remains unknown. Incontinent bitches can be treated successfully with depot preparations of GnRH-analogues and there are differences in plasma gonadotropin levels between continent and incontinent spayed bitches. It is therefore assumed that the supraordinated hormones, GnRH, FSH, and/or LH, have an effect on the urodynamic parameters. In this study, the potential influence of these hormones on the lower urinary tract was investigated by measuring urethral pressure profiles and cystometry. Simultaneously, plasma concentrations in 10 spayed Beagle bitches were determined 5 weeks prior to and 8 weeks after treatment with the GnRH analogue leuprolide. Within 1 week of GnRH analogue administration, plasma FSH and LH levels decreased from 72.5 and 7.7 to 7.75 and 0.72ng/mL, respectively. These plasma gonadotropin levels correspond with those of intact bitches during anoestrus. Urethral pressure profiles indicated that the treatment had no significant effect on maximum urethral closure pressure, functional and total length of the urethra, or area of the closure pressure curve. The data obtained by cystometry regarding mean bladder threshold volume showed a significant increase from 109 to 172mL. The improvement in bladder function after the application of GnRH-application is presumably a direct effect of the GnRH as a relationship between the plasma gonadotropin levels and the urodynamic parameters could not demonstrated.     

Patellofemoral joint compression forces in backward and forward running

Patellofemoral pain (PFP) is a common injury and increased patellofemoral joint compression forces (PFJCF) may aggravate symptoms. Backward running (BR) has been suggested for exercise with reduced PFJCF. The aims of this study were to (1) investigate if BR had reduced peak PFJCF compared to forward running (FR) at the same speed, and (2) if PFJCF was reduced in BR, to investigate which biomechanical parameters explained this. It was hypothesized that (1) PFJCF would be lower in BR, and (2) that this would coincide with a reduced peak knee moment caused by altered ground reaction forces (GRFs). Twenty healthy subjects ran in forward and backward directions at consistent speed. Kinematic and ground reaction force data were collected; inverse dynamic and PFJCF analyses were performed. PFJCF were higher in FR than BR (4.5±1.5; 3.4±1.4BW; p<0.01). The majority of this difference (93.1%) was predicted by increased knee moments in FR compared to BR (157±54; 124±51 Nm; p<0.01). 54.8% of differences in knee moments could be predicted by the magnitude of the GRF (2.3±0.3; 2.4±0.2BW), knee flexion angle (44±6; 41±7) and center of pressure location on the foot (25±11; 12±6%) at time of peak knee moment. Results were not consistent in all subjects. It was concluded that BR had reduced PFJCF compared to FR. This was caused by an increased knee moment, due to differences in magnitude and location of the GRF vector relative to the knee. BR can therefore be used to exercise with decreased PFJCF.     

S-palmitoylation and ubiquitination differentially regulate interferon-induced transmembrane protein 3 (IFITM3)-mediated resistance to influenza virus

The interferon (IFN)-induced transmembrane protein 3 (IFITM3) is a cellular restriction factor that inhibits infection by influenza virus and many other pathogenic viruses. IFITM3 prevents endocytosed virus particles from accessing the host cytoplasm although little is known regarding its regulatory mechanisms. Here we demonstrate that IFITM3 localization to and antiviral remodeling of endolysosomes is differentially regulated by S-palmitoylation and lysine ubiquitination. Although S-palmitoylation enhances IFITM3 membrane affinity and antiviral activity, ubiquitination decreases localization with endolysosomes and decreases antiviral activity. Interestingly, autophagy reportedly induced by IFITM3 expression is also negatively regulated by ubiquitination. However, the canonical ATG5-dependent autophagy pathway is not required for IFITM3 activity, indicating that virus trafficking from endolysosomes to autophagosomes is not a prerequisite for influenza virus restriction. Our characterization of IFITM3 ubiquitination sites also challenges the dual-pass membrane topology predicted for this protein family. We thus evaluated topology by N-linked glycosylation site insertion and protein lipidation mapping in conjunction with cellular fractionation and fluorescence imaging. Based on these studies, we propose that IFITM3 is predominantly an intramembrane protein where both the N and C termini face the cytoplasm. In sum, by characterizing S-palmitoylation and ubiquitination of IFITM3, we have gained a better understanding of the trafficking, activity, and intramembrane topology of this important IFN-induced effector protein.     

Effect of Downed Woody Debris on Small Mammal Anti‐Predator Behavior

Anti‐predator behavior can affect prey growth, reproduction, survival, and generate emergent effects in food webs. Small mammals often lower the cost of predation by altering their behavior in response to shrubs, but the importance of other microhabitat features, such as downed woody debris, for anti‐predator behavior is unknown. We used giving‐up densities to quantify the degree to which downed woody debris alters perceived predation risk by small mammals in southeastern pine forests. We placed 14 foraging trays next to large downed woody debris, shrubs, and in open areas for 12 consecutive nights. Moon illumination, a common indicator of predation risk, led to a similar reduction in small mammal foraging in all three microhabitats (open, downed woody debris, and shrub). Small mammals perceived open microhabitats as riskier than shrub microhabitats, with downed woody debris habitats perceived as being of intermediate risk between shrub and open microhabitats. Despite the presumed benefits of the protective cover of downed woody debris, small mammals may perceive downed woody debris as a relatively risky foraging site in southeastern pine forests where the high diversity and abundance of rodent‐eating snakes may provide a primary predatory threat.     

Large-scale MHC class II genotyping of a wild lemur population by next generation sequencing

The critical role of major histocompatibility complex (MHC) genes in disease resistance, along with their putative function in sexual selection, reproduction and chemical ecology, make them an important genetic system in evolutionary ecology. Studying selective pressures acting on MHC genes in the wild nevertheless requires population-wide genotyping, which has long been challenging because of their extensive polymorphism. Here, we report on large-scale genotyping of the MHC class II loci of the grey mouse lemur (Microcebus murinus) from a wild population in western Madagascar. The second exons from MHC-DRB and -DQB of 772 and 672 individuals were sequenced, respectively, using a 454 sequencing platform, generating more than 800,000 reads. Sequence analysis, through a stepwise variant validation procedure, allowed reliable typing of more than 600 individuals. The quality of our genotyping was evaluated through three independent methods, namely genotyping the same individuals by both cloning and 454 sequencing, running duplicates, and comparing parent–offspring dyads; each displaying very high accuracy. A total of 61 (including 20 new) and 60 (including 53 new) alleles were detected at DRB and DQB genes, respectively. Both loci were non-duplicated, in tight linkage disequilibrium and in Hardy–Weinberg equilibrium, despite the fact that sequence analysis revealed clear evidence of historical selection. Our results highlight the potential of 454 sequencing technology in attempts to investigate patterns of selection shaping MHC variation in contemporary populations. The power of this approach will nevertheless be conditional upon strict quality control of the genotyping data.     

Insights into type IV pilus biogenesis and dynamics from genetic analysis of a C-terminally tagged pilin: a role for O-linked glycosylation

Type IV pili are surface organelles essential for pathogenicity of many Gram-negative bacteria. In Neisseria gonorrhoeae, the major subunit of type IV pili, PilE, is a target of its general O-linked glycosylation system. This system modifies a diverse set of periplasmic and extracellular gonococcal proteins with a variable set of glycans. Here we show that expression of a particular hexa-histidine-tagged PilE was associated with growth arrest. By studying intra- and extragenic suppressors, we found that this phenotype was dependent on pilus assembly and retraction. Based on these results, we developed a sensitive tool to identify factors with subtle effects on pilus dynamics. Using this approach, we found that glycan chain length has differential effects on the growth arrest that appears to be mediated at the level of pilin subunit-subunit interactions and bidirectional remodelling of pilin between its membrane-associated and assembled states. Gonococcal pilin glycosylation thus plays both an intracellular role in pilus dynamics and potential extracellular roles mediated through type IV pili. In addition to demonstrating the effect of glycosylation on pilus dynamics, the study provides a new way of identifying factors with less dramatic effects on processes involved in type IV pilus biogenesis.     

Novel ATP competitive MK2 inhibitors with potent biochemical and cell-based activity throughout the series

Optimization of our previously described pyrrolopiperidone series led to the identification of a new benzamide sub-series, which exhibits consistently high potency in biochemical and cell-based assays throughout the series. Strong inhibition of LPS-induced production of the cytokine TNFα is coupled to the regulation of HSP27 phosphorylation, indicating that the observed cellular effects result from the inhibition of MK2. X-ray crystallographic and computational analyses provide a rationale for the high potency of the series.     

Natural and accelerated recovery from brain damage: experimental and theoretical approaches

The goal of the Caltech group is to gain insight into the processes that occur within the primate nervous system during dexterous reaching and grasping and to see whether natural recovery from local brain damage can be accelerated by artificial means. We will create computational models of the nervous system embodying this insight and explain a variety of clinically observed neurological deficits in human subjects using these models.