Positive feedback in Ras activation by full-length SOS arises from autoinhibition release mechanism.

Signaling through the Ras-MAPK pathway can exhibit switch-like activation, which has been attributed to the underlying positive feedback and bimodality in the activation of RasGDP to RasGTP by SOS. SOS contains both catalytic and allosteric Ras binding sites, and a common assumption is that allosteric activation selectively by RasGTP provides the mechanism of positive feedback. However, recent single-molecule studies have revealed that SOS catalytic rates are independent of the nucleotide state of Ras in the allosteric binding site, raising doubt about this as a positive feedback mechanism. Here, we perform detailed kinetic analyses of receptor-mediated recruitment of full-length SOS to the membrane while simultaneously monitoring its catalytic activation of Ras. These results, along with kinetic modeling, expose the autoinhibition release step in SOS, rather than either recruitment or allosteric activation, as the underlying mechanism giving rise to positive feedback in Ras activation.

Bimodality in Ras signaling originates from processivity of the Ras activator SOS without deterministic bistability.

Ras is a small GTPase that is central to important functional decisions in diverse cell types. An important aspect of Ras signaling is its ability to exhibit bimodal or switch-like activity. We describe the total reconstitution of a receptor-mediated Ras activation-deactivation reaction catalyzed by SOS and p120-RasGAP on supported lipid membrane microarrays. The results reveal a bimodal Ras activation response, which is not a result of deterministic bistability but is rather driven by the distinct processivity of the Ras activator, SOS. Furthermore, the bimodal response is controlled by the condensation state of the scaffold protein, LAT, to which SOS is recruited. Processivity-driven bimodality leads to stochastic bursts of Ras activation even under strongly deactivating conditions. This behavior contrasts deterministic bistability and may be more resistant to pharmacological inhibition.

Bimodality in Ras signaling originates from processivity of the Ras activator SOS without classic kinetic bistability.

Ras is a small GTPase that is central to important functional decisions in diverse cell types. An important aspect of Ras signaling is its ability to exhibit bimodal, or switch-like activity. We describe the total reconstitution of a receptor-mediated Ras activation-deactivation reaction catalyzed by SOS and p120-RasGAP on supported lipid membrane microarrays. The results reveal a bimodal Ras activation response, which is not a result of classic kinetic bistability, but is rather driven by the distinct processivity of the Ras activator, SOS. Furthermore, the bimodal response is controlled by the condensation state of the scaffold protein, LAT, to which SOS is recruited. Processivity-driven bimodality leads to stochastic bursts of Ras activation even under strongly deactivating conditions. This behavior contrasts classic kinetic bistability and is distinctly more resistant to pharmacological inhibition.

A two-component protein condensate of the EGFR cytoplasmic tail and Grb2 regulates Ras activation by SOS at the membrane.

We reconstitute a phosphotyrosine-mediated protein condensation phase transition of the ∼200 residue cytoplasmic tail of the epidermal growth factor receptor (EGFR) and the adaptor protein, Grb2, on a membrane surface. The phase transition depends on phosphorylation of the EGFR tail, which recruits Grb2, and crosslinking through a Grb2-Grb2 binding interface. The Grb2 Y160 residue plays a structurally critical role in the Grb2-Grb2 interaction, and phosphorylation or mutation of Y160 prevents EGFR:Grb2 condensation. By extending the reconstitution experiment to include the guanine nucleotide exchange factor, SOS, and its substrate Ras, we further find that the condensation state of the EGFR tail controls the ability of SOS, recruited via Grb2, to activate Ras. These results identify an EGFR:Grb2 protein condensation phase transition as a regulator of signal propagation from EGFR to the MAPK pathway.

Determining the size and refractive index of homogeneous spherical aerosol particles using Mie resonance spectroscopy.

Methods for determining the size and refractive index of single, homogeneous, micrometer-sized aerosol particles using Mie resonance spectroscopy are studied using measurements from optically trapped particles and light-scattering calculations based on Mie theory. We consider both single-particle broadband light scattering and cavity-enhanced Raman scattering (CERS) and demonstrate that, when resonances observed in either type of spectroscopy are fitted using Mie theory, the accuracy of the best fits are similar. However, broadband measurements can yield more resonances than CERS, thus reducing the uncertainty in the retrieved parameters of best fit and increasing the range of particles that can be characterized. Resonance fitting methods are also compared to methods that fit the entire Mie scattering spectrum. Through calculations, it is shown that measured scattering spectra are sensitive to small changes in how light is collected, while Mie resonance positions are much less sensitive. This means that additional parameters are required to accurately fit entire light-scattering spectra using Mie theory, but these parameters are not needed to accurately determine Mie resonance positions.

MR imaging findings in a dog with lumbar ganglion cysts.

Intraspinal cysts of the L6-L7 and L7-S1 articular process joints in a six-year-old neutered female German Shepherd Dog were diagnosed using magnetic resonance (MR) imaging. Histopathology provided a diagnosis of ganglion cysts. Clinical, laboratory, radiographic and MR imaging findings are described. Briefly, radiographic findings revealed lumbarization of the first sacral vertebra, and fusion of the first caudal vertebra to the sacrum. In addition, spondylosis and articular process osteoarthrosis occurred at L6-L7 and L7-S1. MR imaging revealed multiple, well encapsulated structures ranging in size from 3-10 mm in diameter which were found to arise from the L6-L7 and L7-S1 articular process joints. These cysts had signal intensities that varied from hyperintense to the cerebrospinal fluid (CSF) on T1 weighted images to isointense to CSF on T2 weighted images. Decompressive surgery in conjunction with arthrodesis of these joints resulted in resolution of clinical signs. The dog remained pain-free 1 1/2 years following surgical therapy.

Open-heart correction of tetralogy of Fallot in an acyanotic dog.

Tetralogy of Fallot was diagnosed in an acyanotic 11-month-old dog. Predicted pressure gradient across the pulmonic valve, as assessed by use of continuous wave Doppler echocardiography, was 94.5 mm Hg. Bidirectional shunting was identified by means of selective angiography. Open-heart correction was performed, using a transatrial approach with limited ventriculotomy and cardiopulmonary bypass. The hypertrophied infundibulum was resected, the ventricular septal defect was closed primarily, and a transannular pericardial patch graft was applied. Pressure gradients across the pulmonic valve were 52.9 and 22.8 mm Hg 2 weeks and 4 months after surgery, respectively. Advances in cardiopulmonary bypass, anesthetic management, and use of the transatrial approach may improve the success of open-heart correction of tetralogy of Fallot in dogs.

Deep hypothermic low flow cardiopulmonary bypass in small dogs.

OBJECTIVE: To evaluate the feasibility of and morbidity and mortality associated with cardiopulmonary bypass (CPB) using deep hypothermia and low flow perfusion in adult dogs weighing less than 10 kg. STUDY DESIGN: Prospective, descriptive study. ANIMALS: Two groups of three dogs underwent CPB. Group 1 dogs underwent deep hypothermia (15 to 18 degrees C), 45 minutes of low perfusion flow (20 mL/kg/min) and 1 hour of aortic cross clamp time. In group 2, ultrafiltration of perfusate before discontinuation of bypass was added to the standard treatment. Complete blood counts, serum biochemistry, urine output, ejection fraction, and cardiac output were monitored before and for 7 days after surgery. RESULTS: All dogs were successfully weaned from bypass. Four of six dogs survived, three without major complications. One dog developed and recovered from septic pleuritis. Two dogs died or were euthanatized after surgery because of respiratory or gastrointestinal complications. Minor complications included anemia, hypoproteinemia, and electrolyte disturbances. Transfusion requirements and edema formation were reduced by ultrafiltration. CONCLUSIONS: The observations in this study support the feasibility of low flow hypothermic CPB. Meticulous tissue handling, precise equipment, ultrafiltration, and aggressive postoperative potassium supplementation are recommended for smaller patients. CLINICAL RELEVANCE: Increased sensitivity to adverse sequelae of CPB may be associated with small patient size. Further evaluation is necessary before routine clinical application of low flow hypothermic CPB in this patient population.