Fiber optic probes allow for in situ characterization of cultural heritage objects and analysis of materials that are difficult to access. Positioning these probes is challenging in terms of focal distance, angle of analysis, and stability. Modifications to improve control include stabilizing the probe against a stationary surface, typically mediated by a tripod, or against the artifact itself with a distance regulating sheath that fixes the focal point at the object surface. The first makes the system less portable, while the second eliminates depth profiling capability. An adjustable working distance adapter was created that allows the operator to position a fiber optic probe against the surface of a transparent artifact and move the working distance up to 6 mm into the material while excluding ambient light. The hollow adapter contains no optical fiber, lenses, or windows, so optics are dictated by the fiber optic probe. The tool was created to study the polymeric interlayers in laminated safety glass used in early 20th century aviation and also could be applied to contemporary laminated glass, other multilayer transparent objects, and substances in transparent containers. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
BackgroundBlood platelets undergo a carefully regulated change in shape to serve as the primary mediators of hemostasis and thrombosis. These processes manifest through platelet spreading and aggregation and are dependent on platelet actin cytoskeletal changes orchestrated by the Rho GTPase family member Rac1. To elucidate how Rac1 is regulated in platelets, we captured Rac1-interacting proteins from platelets and identified Rac1-associated proteins by mass spectrometry.FindingsHere, we demonstrate that Rac1 captures the Rac guanine nucleotide exchange factor P-Rex1 from platelet lysates. Western blotting experiments confirmed that P-Rex1 is expressed in platelets and associated with Rac1. To investigate the functional role of platelet P-Rex1, platelets from P-Rex1-/--deficient mice were treated with platelet agonists or exposed to platelet activating surfaces of fibrinogen, collagen and thrombin. Platelets from P-Rex1-/- mice responded to platelet agonists and activating surfaces similarly to wild type platelets.ConclusionsThese findings suggest that P-Rex1 is not required for Rac1-mediated platelet activation and that the GEF activities of P-Rex1 may be more specific to GPCR chemokine receptor mediated processes in immune cells and tumor cells.
A novel, non‐invasive study of goggles, flight helmets, airplane windows, and canopies in Smithsonian collections is the first known large‐scale technical survey of historic aviation plastics and leverages the world's largest air and space collection as evidence of the materials and technologies used to create transparent plastic objects in the early‐20th century. Transparent windows in these artifacts were analyzed with Fourier transform and portable dispersive Raman spectrometers to identify polymers and plasticizers present. The study demonstrates the potential of Raman spectroscopy to objectively and non‐destructively measure historic plastic compositions, including formulations that have become obsolete. Data was interpreted in combination with archival research of historical documents to identify window materials including glass, laminated safety glass, and sheets of plasticized cellulose nitrate, plasticized cellulose acetate, and poly(methyl methacrylate). Results are contextualized into a coherent history of the role transparent plastics played in enclosing airplane cockpits. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
Trauma in pregnancy is a leading cause of poor fetal and obstetric outcomes. Trauma team activation (TTA) criteria include injury with ≥ 20 weeks gestational age (GA). A retrospective analysis was performed on pregnant patients evaluated at a Level 1 trauma center. Patients were characterized by TTA: full, partial, or non-TTA, and TTA criteria independent of pregnancy. Index trauma and delayed delivery hospitalization outcomes were examined. Bivariate analysis, t test, and logistic regression were used when appropriate. From 2010 to 2015, 216 full, 50 partial, and 50 non-TTAs presented. Independent of pregnancy, 79 per cent of patients did not meet the TTA criteria. Fourteen (4%) had a pregnancy-related complication during index hospitalization (eight fetal and two maternal deaths). Nine of ten deaths occurred in patients meeting TTA independent of pregnancy. Delivery complications were greater in the index (52%, 13/25) versus subsequent (5%, 17/155) hospitalizations and were predicted by the respiratory rate ( P = 0.016) and injury severity score ( P < 0.001). Poor delayed delivery outcomes were associated with earlier GA ( P < 0.002) and longer index hospitalization ( P < 0.024). Odds of complication are higher in patients meeting the physiologic and anatomic criteria criteria for TTA versus GA criteria alone, signifying overtriage. Trauma activation protocols should be adapted based on the physiologic and anatomic criteria criteria in pregnant patients.
A major signaling pathway that regulates platelet shape change and reorganization of the cytoskeleton involves the Rho family of GTPases; CDC42 (fillapodia), Rac1 (lamellapodia) and RhoA (focal adhesions). These GTPases are converted from their inactive or GDP-loaded state to the active or GTP-loaded state by a class of enzymes called Guanine Exchange Factors (GEFs). GEFs are a family of multi-domain proteins that contain a GDP-GTP exchange domain (DH-PH) as well as other protein interacting domains that are regulated by the activation of receptors present on the platelet surface. We used an affinity binding technique followed by mass spectroscopy to identify novel Rho family binding GEFs in platelet lysates. Recombinant GST-Rho fusion proteins bound to agarose beads were prepared in the GTP, GDP and nucleotide-free states and incubated with clarified human platelet lysates. Platelet lysate proteins associated with the different GST-Rho preparations were eluted and run on SDS-PAGE. Gel slices were then cut out, trypsin digested and analyzed by Mass Spectroscopy. Platelet GEFs were identified by the presence of a DH-PH domain. Using this technique we found Cool-1 and Cool-2, two closely related GEFs that regulate Rac1 and CDC42 activation in nucleated cells. Cool-1 has not been previously described in platelets. Homodimeric Cool-1 specifically activates CDC42, whereas Cool-2 is capable of activating Rac1 as a homodimer or CDC42 as a monomer. The substrate specificity of Cool-2 is regulated by complex interactions with p21-activated kinases (PAK) and Gb/g proteins. In nucleated cells, Cool-1 and Cool-2 are localized to focal adhesions by the GPCR-kinase-interacting proteins 1 and 2 (GIT1 and GIT2). GIT binding to the Cool proteins has been shown to inhibit their GEF activity. Here we show that Cool-1 is present only in the membrane fraction of resting platelet lysates while Cool-2 is present in both the membrane and cytoplasmic fractions. Upon activation with thrombin, about half the membrane bound Cool-1 and all the membrane bound Cool- 2 migrate to the cytoplasm. Precipitation experiments in thrombin activated platelets with anti-GIT1 and anti-GIT2 antibodies show GIT-1 avidly binds Cool-2 whereas GIT2 binds a small amount of Cool-1. These data suggest a complex mechanism of CDC42 and Rac1 activation in platelets by the Cool proteins. Based on our findings and the current understanding of the regulation of Cool proteins in nucleated cells, we hypothesize that early after thrombin activation, the CDC42 and Rac1 GEF activities of Cool-1 and Cool-2 result in fillapodia (CDC42) and lamellapodia (Rac1) formation. As integrant mediated focal adhesions are formed, the GEF activities of Cool-1 and Cool-2 are inhibited by the scaffolding proteins GIT2 and GIT1, respectively. In this manner, Rac1 and CDC42 mediated fillapodia and lamellapodia formation may be regulated by the extent of integrin engagement and platelet spreading.
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