The Rac-FRET mouse reveals tight spatiotemporal control of Rac activity in primary cells and tissues

The small G protein family Rac has numerous regulators that integrate extracellular signals into tight spatiotemporal maps of its activity to promote specific cell morphologies and responses. Here, we have generated a mouse strain, Rac-FRET, which ubiquitously expresses the Raichu-Rac biosensor. It enables FRET imaging and quantification of Rac activity in live tissues and primary cells without affecting cell properties and responses. We assessed Rac activity in chemotaxing Rac-FRET neutrophils and found enrichment in leading-edge protrusions and unexpected longitudinal shifts and oscillations during protruding and stalling phases of migration. We monitored Rac activity in normal or disease states of intestinal, liver, mammary, pancreatic, and skin tissue, in response to stimulation or inhibition and upon genetic manipulation of upstream regulators, revealing unexpected insights into Rac signaling during disease development. The Rac-FRET strain is a resource that promises to fundamentally advance our understanding of Rac-dependent responses in primary cells and native environments

Drug Discovery of Novel Targeted Therapeutics for Metastatic Breast Cancer

Metastatic disease is the primary cause of breast cancer mortality, due to the lack of effective therapy. The Rho GTPase Rac is integral for the promotion of cancer cell migration/invasion, proliferation, and survival. Since metastatic breast cancers often overexpress or exhibit high Rac activity, inhibition of Rac is a viable strategy against metastatic cancer. Recently, we characterized EHop-016, a small molecule that inhibits Rac activity of metastatic breast cancer cells more efficiently than previously available Rac inhibitors (IC50 of 1µM). EHop-016 inhibits the activity of the Rac downstream effector p21 activated kinase and cell migration of metastatic breast cancer cells. We also reported that EHop-016 at ≥ 25mg/kg body weight significantly reduced tumor growth and metastasis in mice. However, our recent pharmacokinetic study of EHop-016 in a mouse model demonstrated that the bioavailability of Ehop-016 needs to be improved for pharmacological development. The hypothesis is that improvement of the EHop-016 structure will provide probes with increased potency against Rac and, therefore, increased bioavailability. Several Ehop-016 derivatives have been tested for their effects on breast cancer cell viability using the MTT assay. We found one compound, HV-107, which at concentrations ≥1µM inhibits the viability of metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-435 by 45%. The effects of HV-107 on the inhibition of Rac activation were tested by pulldown assays. At 250nM, HV-107 inhibits Rac activation by 55% in MDA-MB-231 and MDA-MB-435 cells. Taken together, our findings suggest HV-107 has potential as an anti-metastatic agent and should, therefore, be further characterized

Reciprocal regulation of PKA and rac signaling

Activated G protein-coupled receptors (GPCRs) and receptor tyrosine kinases relay extracellular signals through spatial and temporal controlled kinase and GTPase entities. These enzymes are coordinated by multifunctional scaffolding proteins for precise intracellular signal processing. The cAMP-dependent protein kinase A (PKA) is the prime example for compartmentalized signal transmission downstream of distinct GPCRs. A-kinase anchoring proteins tether PKA to specific intracellular sites to ensure precision and directionality of PKA phosphorylation events. Here, we show that the Rho-GTPase Rac contains A-kinase anchoring protein properties and forms a dynamic cellular protein complex with PKA. The formation of this transient core complex depends on binary interactions with PKA subunits, cAMP levels and cellular GTP-loading accounting for bidirectional consequences on PKA and Rac downstream signaling. We show that GTP-Rac stabilizes the inactive PKA holoenzyme. However, β-adrenergic receptor-mediated activation of GTP-Rac–bound PKA routes signals to the Raf-Mek-Erk cascade, which is critically implicated in cell proliferation. We describe a further mechanism of how cAMP enhances nuclear Erk1/2 signaling: It emanates from transphosphorylation of p21-activated kinases in their evolutionary conserved kinase-activation loop through GTP-Rac compartmentalized PKA activities. Sole transphosphorylation of p21-activated kinases is not sufficient to activate Erk1/2. It requires complex formation of both kinases with GTP-Rac1 to unleash cAMP-PKA–boosted activation of Raf-Mek-Erk. Consequently GTP-Rac functions as a dual kinase-tuning scaffold that favors the PKA holoenzyme and contributes to potentiate Erk1/2 signaling. Our findings offer additional mechanistic insights how β-adrenergic receptor-controlled PKA activities enhance GTP-Rac–mediated activation of nuclear Erk1/2 signaling

Activation of Rac-1 and RhoA contributes to podocyte injury in chronic kidney disease

Rho-family GTPases like RhoA and Rac-1 are potent regulators of cellular signaling that control gene expression, migration and inflammation. Activation of Rho-GTPases has been linked to podocyte dysfunction, a feature of chronic kidney diseases (CKD). We investigated the effect of Rac-1 and Rho kinase (ROCK) inhibition on progressive renal failure in mice and studied the underlying mechanisms in podocytes. SV129 mice were subjected to 5/6-nephrectomy which resulted in arterial hypertension and albuminuria. Subgroups of animals were treated with the Rac-1 inhibitor EHT1846, the ROCK inhibitor SAR407899 and the ACE inhibitor Ramipril. Only Ramipril reduced hypertension. In contrast, all inhibitors markedly attenuated albumin excretion as well as glomerular and tubulo-interstitial damage. The combination of SAR407899 and Ramipril was more effective in preventing albuminuria than Ramipril alone. To study the involved mechanisms, podocytes were cultured from SV129 mice and exposed to static stretch in the Flexcell device. This activated RhoA and Rac-1 and led via TGFβ to apoptosis and a switch of the cells into a more mesenchymal phenotype, as evident from loss of WT-1 and nephrin and induction of α-SMA and fibronectin expression. Rac-1 and ROCK inhibition as well as blockade of TGFβ dramatically attenuated all these responses. This suggests that Rac-1 and RhoA are mediators of podocyte dysfunction in CKD. Inhibition of Rho-GTPases may be a novel approach for the treatment of CKD

Can a web-based community of practice be established and operated to lead falls prevention activity in residential care?

The aims of this study were to evaluate establishing and operating a web-based community of practice (CoP) to lead falls prevention in a residential aged care (RAC) setting. A mixed methods evaluation was conducted in two phases using a survey and transcripts from interactive electronic sources. Nurses and allied health staff (n = 20) with an interest in falls prevention representing 13 sites of an RAC organization participated. In Phase 1, the CoP was developed, and the establishment of its structure and composition was evaluated using determinants of success reported in the literature. In Phase 2, all participants interacted using the web, but frequency of engagement by any participant was low. Participatory barriers, including competing demands from other tasks and low levels of knowledge about information communication technology (ICT) applications, were identified by CoP members. A web-based CoP can be established and operated across multiple RAC sites if RAC management support dedicated time for web-based participation and staff are given web-based training

Notes on large angle crossing graphs

A graph G is an a-angle crossing (aAC) graph if every pair of crossing edges in G intersect at an angle of at least a. The concept of right angle crossing (RAC) graphs (a=Pi/2) was recently introduced by Didimo et. al. It was shown that any RAC graph with n vertices has at most 4n-10 edges and that there are infinitely many values of n for which there exists a RAC graph with n vertices and 4n-10 edges. In this paper, we give upper and lower bounds for the number of edges in aAC graphs for all 0 < a < Pi/2

Rapid identification and differentiation of the vaccine strain Rac H from EHV 1 field isolates using a non-radioactive DNA probe

A method for rapid differentiation between the EHV 1 live vaccine strain Rac H and field isolates is described. Total DNA was isolated from virus-infected small scale cell cultures. DNA fragments digested with restriction endonuclease BamHI were separated, transfered and immobilized on filter membranes. A Digoxigenin-labeled probe derived from EHV 1 was used for hybridization. This probe hybridized specifically to sequences of the inverted terminal repeat region which in case of Rac H include a deletion of 0.8 kb. By comparing the different migration patterns after blot hybridization it could be shown that in 65 isolates from cases of abortion the live vaccine strain Rac H was not involve

Dobutamine stress MRI in pulmonary hypertension: relationships between stress pulmonary artery relative area change, RV performance, and 10-year survival

In pulmonary hypertension (PH), right ventricular (RV) performance determines survival. Pulmonary artery (PA) stiffening is an important biomechanical event in PH and also predicts survival based on the PA relative area change (RAC) measured at rest using magnetic resonance imaging (MRI). In this exploratory study, we sought to generate novel hypotheses regarding the influence of stress RAC on PH prognosis and the interaction between PA stiffening, RV performance and survival. Fifteen PH patients underwent dobutamine stress-MRI (ds-MRI) and right heart catheterization. RACREST, RACSTRESS, and ΔRAC (RAC STRESS – RAC REST) were correlated against resting invasive hemodynamics and ds-MRI data regarding RV performance and RV-PA coupling efficiency (n’vv [RV stroke volume/RV end-systolic volume]). The impact of RAC, RV data, and n’vv on ten-year survival were determined using Kaplan–Meier analysis. PH patients with a low ΔRAC (&lt;−2.6%) had a worse long-term survival (log-rank P = 0.045, HR for death = 4.46 [95% CI = 1.08–24.5]) than those with ΔRAC ≥ −2.6%. Given the small sample, these data should be interpreted with caution; however, low ΔRAC was associated with an increase in stress diastolic PA area indicating proximal PA stiffening. Associations of borderline significance were observed between low RACSTRESS and low n’vvSTRESS, Δη’VV, and ΔRVEF. Further studies are required to validate the potential prognostic impact of ΔRAC and the biomechanics potentially connecting low ΔRAC to shorter survival. Such studies may facilitate development of novel PH therapies targeted to the proximal PA

Compact Drawings of 1-Planar Graphs with Right-Angle Crossings and Few Bends

We study the following classes of beyond-planar graphs: 1-planar, IC-planar, and NIC-planar graphs. These are the graphs that admit a 1-planar, IC-planar, and NIC-planar drawing, respectively. A drawing of a graph is 1-planar if every edge is crossed at most once. A 1-planar drawing is IC-planar if no two pairs of crossing edges share a vertex. A 1-planar drawing is NIC-planar if no two pairs of crossing edges share two vertices. We study the relations of these beyond-planar graph classes (beyond-planar graphs is a collective term for the primary attempts to generalize the planar graphs) to right-angle crossing (RAC) graphs that admit compact drawings on the grid with few bends. We present four drawing algorithms that preserve the given embeddings. First, we show that every $n$-vertex NIC-planar graph admits a NIC-planar RAC drawing with at most one bend per edge on a grid of size $O(n) \times O(n)$. Then, we show that every $n$-vertex 1-planar graph admits a 1-planar RAC drawing with at most two bends per edge on a grid of size $O(n^3) \times O(n^3)$. Finally, we make two known algorithms embedding-preserving; for drawing 1-planar RAC graphs with at most one bend per edge and for drawing IC-planar RAC graphs straight-line