Evidence that the LRRK2 ROC domain Parkinson's disease‐associated mutants A1442P and R1441C exhibit increased intracellular degradation

Mutations in the leucine‐rich repeat kinase 2 (lrrk2) gene are the leading genetic cause of Parkinson's disease (PD). In characterizing the novel ROC domain mutant A1442P, we compared its steady‐state protein levels, propensity to aggregate, and toxicity with the pathogenic R1441C mutant and wild‐type (WT) LRRK2. Mutant (R1441C and A1442P) and WT LRRK2 fused to green fluorescent protein (GFP) and FLAG were transiently expressed in HEK293 cells using plasmid constructs. Western analysis and fluorescence microscopy consistently demonstrated lower mutant LRRK2 protein levels compared with WT. A time‐course expression study using flow cytometry showed that WT LRRK2 expression increased initially but then plateaued by 72 hr. Conversely, R1441C and A1442P mutant expression attained 85% and 74% of WT levels at 24 hr but fell to 68% and 55% of WT levels by 72 hr, respectively. We found that proteasome inhibition markedly increased mutant LRRK2 to levels approaching those of WT. Taken together, our findings reveal increased intracellular degradation for both mutants. Furthermore, the impact of mutant and WT LRRK2 expression on HEK293 cell viability was assessed under normative and oxidative (hydrogen peroxide) conditions and found not to differ. Expression of WT and mutant LRRK2 protein gave rise to intracellular aggregates of similar appearance and cellular localization. In summary, we provide evidence that the novel A1442P mutant and the previously investigated R1441C pathogenic mutant exhibit increased intracellular degradation, a property reportedly demonstrated for the pathogenic LRRK2 kinase domain mutant I2020T. © 2013 Wiley Periodicals, Inc.     

Assessment of cyclic changes of microvessels in ovine ovaries using Sonovue contrast-enhanced ultrasound

Our objective was to detect variations of ovarian vascularization that occur in the estrus cycle of the ewe using an IV contrast agent. Five ewes were investigated using contrast-enhanced power Doppler after Sonovue injection at day 0, 3, 5, 10 and 13 of the cycle in two successive estrus cycles. Transvaginal ultrasound monitoring of each ewe was performed after a dose of Sonovue. Parameters derived from the time-intensity curves were compared. Quantification of the enhancement and wash-in period parameters changed significantly between ovaries and between the follicular and luteal periods of the cycle. Uptake time, wash-out time, total time of enhancement and area under the curve were the parameters with the smallest variation between ovaries. Wash-out time and area under the curve are two contrast parameters that did not change with cyclic changes. Thus, using these parameters in premenopausal women will allow more accurate detection of vascular modifications that may be associated with ovarian cancer.     

What is the Safest Sprint Starting Position for American Football Players?

The main objective of this study was to perform a biomechanical analysis of three different sprint start patterns to determine the safest position in term of neck injury and Sport-Related Concussion (SRC). The second objective was to collect data on the learning process effect between football players and non-players. Three different sprint initial positions adopted by football players were studied (i.e., 4-, 3- and 2-point positions). Twenty five young healthy males, including 12 football players, participated to this study. A stereophotogrammetric system (i.e., Vicon) was used to record motion patterns and body segments positions. Various measurements related to head and trunk orientation, and player field-of-view were obtained (e.g., head height, trunk bending, time to reach upright position, head speed (vertical direction) and body speed (horizontal direction)). Learning process was found to have no influence on studied parameters. Head redress is also delayed when adopting a 4-point position leading to a reduce field-of-view during the start and increasing therefore the probability of collision. Concerning the three different positions, the 4-point position seems to be the more dangerous because leading to higher kinetic energy than the 2- and 3-point start positions. This study proposes a first biomechanical approach to understand risk/benefit balance for athletes for those three different start positions. Results suggested that the 4-point position is the most risky for football players.

Key points

• Motion analysis and biomechanical analysis of the initial start position of the sprint could be used to increase the safety of the football players.
• Analysis of kinematic and trajectory of the head and the time to reach the upright position could be used to determine whether or not a player can return to play after concussion.
• A balance needs to be found between player’s safety (2-point start) and speed (4-point start).

Key words: Sports, sports medicine, brain concussion, biomechanics     

Surface structure and mechanical properties of impaction-modified Y-TZP

Objectives

The objectives of the study were to describe the surface structure and the chemical surface composition of Y-TZP ceramics produced by using the modified additive technique and to evaluate the flexural strength of Y-TZP with or without surface modification and with different pretreatments: etching before or after sintering combined with or without an adhesive cement system.

Methods

Y-TZP discs were used for surface analysis (n = 48) and for biaxial flexural strength testing (n = 200). The specimens were divided into groups depending on the cementation surface of Y-TZP: unmodified, sandblasted or glass-modified Y-TZP surfaces, and according to the production process: etching before or after sintering.

Results

The surface structure and the chemical composition of glass-modified Y-TZP differ; a rougher surface and phase transformation was identified compared to unmodified Y-TZP.The unmodified Y-TZP groups showed significantly higher flexural strength compared to the glass-modified groups (p < 0.001) and showed increased flexural strength after sandblasting (p < 0.001). Furthermore, by adding cement to the surface, the value increased even further in comparison with the sandblasted non-cemented specimens (p < 0.01). After thermocycling, however, the cement layer on the unmodified and the sandblasted surfaces had air pockets and regions with loose cement.

Significance

A rougher surface structure, superficial glass remnants and a higher content of m-phase was present in the cementation surface of glass-modified Y-TZP. The glass modification creates a bondable cementation surface that is durable. By etching the glass-modified Y-TZP before sintering, a more homogenous surface is created compared to one that is etched after sintering.