Poxviral Protein A52 Stimulates p38 Mitogen-activated Protein Kinase (MAPK) Activation by Causing Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) Self-association Leading to Transforming Growth Factor β-activated Kinase 1 (TAK1) Recruitment

Vaccinia virus encodes a number of proteins that inhibit and manipulate innate immune signaling pathways that also have a role in virulence. These include A52, a protein shown to inhibit IL-1- and Toll-like receptor-stimulated NFκB activation, via interaction with interleukin-1 receptor-associated kinase 2 (IRAK2). Interestingly, A52 was also found to activate p38 MAPK and thus enhance Toll-like receptor-dependent IL-10 induction, which was TRAF6-dependent, but the manner in which A52 manipulates TRAF6 to stimulate p38 activation was unclear. Here, we show that A52 has a non-canonical TRAF6-binding motif that is essential for TRAF6 binding and p38 activation but dispensable for NFκB inhibition and IRAK2 interaction. Wild-type A52, but not a mutant defective in p38 activation and TRAF6 binding (F154A), caused TRAF6 oligomerization and subsequent TRAF6-TAK1 association. The crystal structure of A52 shows that it adopts a Bcl2-like fold and exists as a dimer in solution. Residue Met-65 was identified as being located in the A52 dimer interface, and consistent with that, A52-M65E was impaired in its ability to dimerize. A52-M65E although capable of interacting with TRAF6, was unable to cause either TRAF6 self-association, induce the TRAF6-TAK1 association, or activate p38 MAPK. The results suggest that an A52 dimer causes TRAF6 self-association, leading to TAK1 recruitment and p38 activation. This reveals a molecular mechanism whereby poxviruses manipulate TRAF6 to activate MAPKs (which can be proviral) without stimulating antiviral NFκB activation.     

Effect of the Fatigue Induced by a 110-km Ultramarathon on Tibial Impact Acceleration and Lower Leg Kinematics

Ultramarathon runners are exposed to a high number of impact shocks and to severe neuromuscular fatigue. Runners may manage mechanical stress and muscle fatigue by changing their running kinematics. Our purposes were to study (i) the effects of a 110-km mountain ultramarathon (MUM) on tibial shock acceleration and lower limb kinematics, and (ii) whether kinematic changes are modulated according to the severity of neuromuscular fatigue. Twenty-three runners participated in the study. Pre- and post-MUM, neuromuscular tests were performed to assess knee extensor (KE) and plantar flexor (PF) central and peripheral fatigue, and a treadmill running bouts was completed during which step frequency, peak acceleration, median frequency and impact frequency content were measured from tibial acceleration, as well as foot-to-treadmill, tibia-to-treadmill, and ankle flexion angles at initial contact, and ankle range of motion using video analysis. Large neuromuscular fatigue, including peripheral changes and deficits in voluntary activation, was observed in KE and PF. MVC decrements of ~35% for KE and of ~28% for PF were noted. Among biomechanical variables, step frequency increased by ~2.7% and the ankle range of motion decreased by ~4.1% post-MUM. Runners adopting a non rearfoot strike pre-MUM adopted a less plantarflexed foot strike pattern post-MUM while those adopting a rearfoot strike pre-MUM tended to adopt a less dorsiflexed foot strike pattern post-MUM. Positive correlations were observed between percent changes in peripheral PF fatigue and the ankle range of motion. Peripheral PF fatigue was also significantly correlated to both percent changes in step frequency and the ankle angle at contact. This study suggests that in a fatigued state, ultratrail runners use compensatory/protective adjustments leading to a flatter foot landing and this is done in a fatigue dose-dependent manner. This strategy may aim at minimizing the overall load applied to the musculoskeletal system, including impact shock and muscle stretch.     

Identification of individual sex-factor DNA strands and their replication during conjugation in thermosensitive DNA mutants of Escherichia coli

Covalent circular sex-factor DNA has been isolated from donor and recipient cells during the conjugation of normal and temperature-sensitive DNA mutants of Escherichia coli. Single strands of sex-factor DNA were centrifuged in cesium chloride-poly(U,G) gradients to give two components that have been identified by annealing experiments as the separated complementary strands. When matings are performed with either DNA temperature-sensitive donor or recipient cells, the inhibition of vegetative DNA synthesis at the restrictive temperature does not interfere with transfer and circularization of the sex-factor DNA. If DNA is isolated from temperature-sensitive donor cells mated at the restrictive temperature, a specific stimulation of sex-factor DNA synthesis can be demonstrated. By separating the complementary strands of the sex-factor in a cesium chloride-poly (U,G) gradient, this DNA synthesis has been found to be asymmetric. The sex-factor DNA strand which is synthesized in the donor has the same polarity as the strand which is transferred to the recipient.     

A statistical human rib cage geometry model accounting for variations by age,sex, stature and body mass index

In this study, we developed a statistical rib cage geometry model accounting for variations by age, sex, stature and body mass index (BMI). Thorax CT scans were obtained from 89 subjects approximately evenly distributed among 8 age groups and both sexes. Threshold-based CT image segmentation was performed to extract the rib geometries, and a total of 464 landmarks on the left side of each subject?s ribcage were collected to describe the size and shape of the rib cage as well as the cross-sectional geometry of each rib. Principal component analysis and multivariate regression analysis were conducted to predict rib cage geometry as a function of age, sex, stature, and BMI, all of which showed strong effects on rib cage geometry. Except for BMI, all parameters also showed significant effects on rib cross-sectional area using a linear mixed model. This statistical rib cage geometry model can serve as a geometric basis for developing a parametric human thorax finite element model for quantifying effects from different human attributes on thoracic injury risks.     

An injury risk curve for the hip for use in frontal impact crash testing

To facilitate the assessment of hip injury risk in frontal motor-vehicle crashes, an injury risk curve that relates peak force transmitted to the hip to the probability of hip fracture was developed by using survival analysis to fit a lognormal distribution to a recently published dataset of hip fracture forces. This distribution was parameterized to account for the effect of subject stature, which was the only subject characteristic found to significantly affect hip fracture force (X²(1)=6.03, p=0.014). The distribution was further parameterized to account for the effects of hip flexion and abduction from a standard driving posture on hip fracture force using relationships between mean hip fracture force and hip flexion/abduction reported in the literature. The resulting parametric distribution was used to define relationships between force applied to the hip and the risk of hip fracture for the statures associated with the small female, midsize male, and large male crash-test dummies, thus allowing these dummies to assess hip fracture/dislocation risk in frontal crashes, provided that such dummies are sufficiently biofidelic. For the midsize male crash test dummy, a 50% risk of hip fracture was associated with a force of 6.00 kN. For the small female and large male dummies, a 50% risk of hip fracture was associated with forces of 4.46 and 6.73 kN, respectively.     

Whole blood,flow-chamber studies in real-time indicate a biphasic role for thymosin β-4 in platelet adhesion

Background

Thymosin beta 4 (Tβ₄) is a major actin sequestering peptide present in most mammalian cells. It also acts as an anti-inflammatory agent and promotes corneal wound healing.

Methods

In the present study, we constructed a four channel cylindrical flow chambers out of polydimethylsiloxane (PDMS) on microscope coverslips. The platelet-binding proteins–fibrinogen and collagen–were immobilized onto the middle ~ 25% of the inner cylindrical surface. The flow method introduced here was employed to determine the effect of Tβ₄, on the deposition of ADP-activated platelets onto fibrinogen cross-linked flow chambers.

Results

The binding data from the flow chambers indicated that the both the rate constant of platelet deposition (average: 0.026 ± 0.0015 s^− 1, corresponding to a half-life of 26.7 s) and the total number of deposited platelets were independent of the platelet binding protein and the activating agent. Our results show that low concentrations of Tβ₄ (0.2 μM to 0.5 μM) increased both the rate constant of platelet deposition by ~ 1.5-fold (i.e. half-life decreased from 26.7 s to 17.6 s) and the total number of deposited platelets by ~ 3-fold. However at higher concentrations (> 1 μM) the Tβ₄-potentiating effect was diminished to near control levels. Tβ₄ did interact with fibrinogen with an estimated K_D of ~ 126 ± 18 nM or 66 ± 20 nM under equilibrium or flow, respectively.

Conclusion

These results suggest that Tβ₄ could potentially increase the affinity of platelet receptors for their ligands thus promoting platelet deposition. Tβ₄ could also bind to fibrinogen and as its concentration increased would prevent platelet–fibrinogen interactions resulting in the attenuation of platelet deposition.

General significance

This work suggests that Tβ₄ might have a dual role in platelet function.     

Antibody elbow angles are influenced by their light chain class

We have examined the elbow angles for 365 different Fab fragments, and observe that Fabs with lambda light chains have adopted a wider range of elbow angles than their kappa chain counterparts, and that the lambda light chain Fabs are frequently found with very large (>195 degrees ) elbow angles. This apparent hyperflexibility of lambda chain Fabs may be due to an insertion in their switch region, which is one residue longer than in kappa chains, with glycine occurring most frequently at the insertion position. A new, web-based computer program that was used to calculate the Fab elbow angles is described.