Comparing the Affinity of GTPase-binding Proteins using Competition Assays

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately

Ultrasonic Stimulation of Mouse Skin Reverses the Healing Delays in Diabetes and Aging by Activation of Rac1

Chronic skin-healing defects are one of the leading challenges to lifelong well-being, affecting 2–5% of populations. Chronic wound formation is linked to age and diabetes and frequently leads to major limb amputation. Here we identify a strategy to reverse fibroblast senescence and improve healing rates. In healthy skin, fibronectin activates Rac1 in fibroblasts, causing migration into the wound bed, and driving wound contraction. We discover that mechanical stimulation of the skin with ultrasound can overturn healing defects by activating a calcium/CamKinaseII/Tiam1/Rac1 pathway that substitutes for fibronectin-dependent signaling and promotes fibroblast migration. Treatment of diabetic and aged mice recruits fibroblasts to the wound bed and reduces healing times by 30%, restoring healing rates to those observed in young, healthy animals. Ultrasound treatment is equally effective in rescuing the healing defects of animals lacking fibronectin receptors, and can be blocked by pharmacological inhibition of the CamKinaseII pathway. Finally, we discover that the migration defects of fibroblasts from human venous leg ulcer patients can be reversed by ultrasound, demonstrating that the approach is applicable to human chronic samples. By demonstrating that this alternative Rac1 pathway can substitute for that normally operating in the skin, we identify future opportunities for management of chronic wounds

Theory of Current-Induced Magnetization Precession

We solve appropriate drift-diffusion and Landau-Lifshitz-Gilbert equations to demonstrate that unpolarized current flow from a non-magnet into a ferromagnet can produce a precession-type instability of the magnetization. The fundamental origin of the instability is the difference in conductivity between majority spins and minority spins in the ferromagnet. This leads to spin accumulation and spin currents that carry angular momentum across the interface. The component of this angular momentum perpendicular to the magnetization drives precessional motion that is opposed by Gilbert damping. Neglecting magnetic anisotropy and magnetostatics, our approximate analytic and exact numerical solutions using realistic values for the material parameters show (for both semi-infinite and thin film geometries) that a linear instability occurs when both the current density and the excitation wave vector parallel to the interface are neither too small nor too large. For many aspects of the problem, the variation of the magnetization in the direction of the current flows makes an important contribution.Comment: Submitted to Physical Review

Co-infections and multiple stressors in fish

Fish are typically exposed to multiple physical, chemical and biological stressors. The cumulative impact of co-infections between parasites, bacteria, viruses and (a)biotic environmental pressures may trigger complex interactions, eliciting different pathological and immunological outcomes than those classically assessed. New cross-disciplinary studies attempt to measure the impact of environmental stressors in modulating the host response to pathogens. Scientific advances are needed to reduce pressure on natural populations, improve fish stock management, and to design more efficient diagnostic tools or vaccination strategies. An EAFP-promoted workshop, held on 10th September 2019 in Porto, Portugal, was dedicated to sharing research experiences on the interaction between heterogenous pathogens and multiple stressors in fish. The workshop involved around 200 attendants, opened by a keynote talk (Fast), and followed by a further twelve oral presentations, including three in the format of lash poster presentations. Contributions illustrated cross-disciplinary approaches to study complex host-pathogen and stressors interactions

A self-consistent treatment of non-equilibrium spin torques in magnetic multilayers

It is known that the transfer of spin angular momenta between current carriers and local moments occurs near the interface of magnetic layers when their moments are non-collinear. However, to determine the magnitude of the transfer, one should calculate the spin transport properties far beyond the interface regions. Based on the spin diffusion equation, we present a self-consistent approach to evaluate the spin torque for a number of layered structures. One of the salient features is that the longitudinal and transverse components of spin accumulations are inter-twined from one layer to the next, and thus, the spin torque could be significantly amplified with respect to treatments which concentrate solely on the transport at the interface due to the presence of the much longer longitudinal spin diffusion length. We conclude that bare spin currents do not properly estimate the spin angular momentum transferred between to the magnetic background; the spin transfer that occurs at interfaces should be self-consistently determined by embedding it in our globally diffuse transport calculations.Comment: 21 pages, 6 figure

The β3-integrin endothelial adhesome regulates microtubule-dependent cell migration

Integrin β3 is seen as a key anti-angiogenic target for cancer treatment due to its expression on neovasculature, but the role it plays in the process is complex; whether it is pro- or anti-angiogenic depends on the context in which it is expressed. To understand precisely β3's role in regulating integrin adhesion complexes in endothelial cells, we characterised, by mass spectrometry, the β3-dependent adhesome. We show that depletion of β3-integrin in this cell type leads to changes in microtubule behaviour that control cell migration. β3-integrin regulates microtubule stability in endothelial cells through Rcc2/Anxa2-driven control of active Rac1 localisation. Our findings reveal that angiogenic processes, both in vitro and in vivo, are more sensitive to microtubule targeting agents when β3-integrin levels are reduced

Breathing Current Domains in Globally Coupled Electrochemical Systems: A Comparison with a Semiconductor Model

Spatio-temporal bifurcations and complex dynamics in globally coupled intrinsically bistable electrochemical systems with an S-shaped current-voltage characteristic under galvanostatic control are studied theoretically on a one-dimensional domain. The results are compared with the dynamics and the bifurcation scenarios occurring in a closely related model which describes pattern formation in semiconductors. Under galvanostatic control both systems are unstable with respect to the formation of stationary large amplitude current domains. The current domains as well as the homogeneous steady state exhibit oscillatory instabilities for slow dynamics of the potential drop across the double layer, or across the semiconductor device, respectively. The interplay of the different instabilities leads to complex spatio-temporal behavior. We find breathing current domains and chaotic spatio-temporal dynamics in the electrochemical system. Comparing these findings with the results obtained earlier for the semiconductor system, we outline bifurcation scenarios leading to complex dynamics in globally coupled bistable systems with subcritical spatial bifurcations.Comment: 13 pages, 11 figures, 70 references, RevTex4 accepted by PRE http://pre.aps.or

A Historiometric Examination of Machiavellianism and a New Taxonomy of Leadership

Although researchers have extensively examined the relationship between charismatic leadership and Machiavellianism (Deluga, 2001; Gardner & Avolio, 1995; House & Howell, 1992), there has been a lack of investigation of Machiavellianism in relation to alternative forms of outstanding leadership. Thus, the purpose of this investigation was to examine the relationship between Machiavellianism and a new taxonomy of outstanding leadership comprised of charismatic, ideological, and pragmatic leaders. Using an historiometric approach, raters assessed Machiavellianism via the communications of 120 outstanding leaders in organizations across the domains of business, political, military, and religious institutions. Academic biographies were used to assess twelve general performance measures as well as twelve general controls and five communication specific controls. The results indicated that differing levels of Machiavellianism is evidenced across the differing leader types as well as differing leader orientation. Additionally, Machiavellianism appears negatively related to performance, though less so when type and orientation are taken into account.Yeshttps://us.sagepub.com/en-us/nam/manuscript-submission-guideline