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Considerable variation in the use and duration of antiplatelet medications during the perioperative and postoperative care of patients undergoing coronary artery bypass grafting (CABG) reflects the limited number of studies focused directly on these patients as well as the variation in the results reported. In this review we highlight the incidence and mechanisms of graft closure as well as the evidence in support of antiplatelet therapy that is balanced by the impact of antiplatelet therapy on the risk of bleeding to provide recommendations for the use of this therapy in patients undergoing CABG. Low-dose acetylsalicylic acid (ASA; ≤ 160 mg daily) reduces the incidence of perioperative myocardial infarction, acute renal injury, and mortality without increasing the risk of bleeding and so is recommended both before and after CABG. The use of dual antiplatelet therapy with ASA plus a P2Y12 antagonist adds a greater risk of bleeding. While additional studies are required, we can make the following recommendations: because of increased bleeding and mortality when patients are treated with clopidogrel preoperatively, CABG should be delayed for five days. Because of increased bleeding when patients are treated with ticagrelor preoperatively, CABG should be delayed for three days. Because of increased bleeding when patients are treated with prasugrel preopera-tively, CABG should be delayed for seven days. For patients who had a coronary stent placed preoperatively or had an acute coronary syndrome preoperatively, resumption of therapy with their P2Y12 antagonist postoperatively for 12 months reduces the subsequent incidence of cardiovascular events.

Mount Cleveland volcano (1730 m) is one of the most active volcanoes in the Aleutian arc, Alaska, but heightened activity is rarely accompanied by geophysical signals, which makes interpretation of the activity difficult. In this study, we combine volcanic gas emissions measured for the first time in August 2015 with longer-term measurements of thermal output and lava extrusion rates between 2011 and 2015 calculated from MODIS satellite data with the aim to develop a better understanding of the nature of volcanic activity at Mount Cleveland. Degassing measurements were made in the month following two explosive events (21 July and 7 August 2015) and during a period of new dome growth in the summit crater. SO2 emission rates ranged from 400 to 860 t d− 1 and CO2/SO2 ratios were

Platelet expression of FcγRIIa was quantified after myocardial infarction (MI) and we found that patients with high platelet FcγRIIa expression (>11,000/platelet) had a fourfold greater risk of subsequent MI, stroke, and death. This analysis of the original cohort of 197 patients was designed to determine whether platelet expression of FcγRIIa could be used in combination with clinical risk scores (GRACE [Global Registry of Acute Coronary Events] and DAPT [Dual Antiplatelet Therapy]) to refine cardiovascular risk assessment. Platelet expression of FcγRIIa quantified with the use of flow cytometry was broadly distributed in patients stratified into high and low risk groups based on clinical risk scores. In patients identified as high risk by the GRACE score, 62% had high platelet FcγRIIa expression. Similarly, in patients identified as high risk by DAPT, 55% had high platelet FcγRIIa expression. High platelet FcγRIIa expression discriminated high and low risk cohorts in patients with high cardiovascular risk defined by either the GRACE score (high platelet FcγRIIa 18.9% vs low platelet FcγRIIa 0%; odds ratio = 15.7, p = 0.06) or the DAPT score (high platelet FcγRIIa 15.4% vs low platelet FcγRIIa 3.7%; odds ratio = 5.6, p = 0.03) assessment. Platelet expression of FcγRIIa merits additional study to determine whether low platelet FcγRIIa expression can be used to guide early transition to aspirin monotherapy and high platelet FcγRIIa expression can be used to guide continuation of DAPT.

FcγRIIa amplifies platelet activation and greater platelet expression of FcγRIIa identifies patients at greater risk of subsequent cardiovascular events. Thus, platelet expression of FcγRIIa may be useful to guide therapy. Because platelet function tests are impacted by preparative procedures and substantial intra-individual variability, we examined the impact of these factors on platelet expression of FcγRIIa in blood from healthy subjects and in patients after myocardial infarction (MI). Platelet expression of FcγRIIa was quantified with the use of flow cytometry. Blood was taken from healthy subjects and 114 patients after a MI in whom platelet expression of FcγRIIa was quantified before discharge and at 6 ± 1 months. Neither anticoagulants nor the antiplatelet agent cangrelor changed platelet expression of FcγRIIa. Intra-individual variation in platelet FcγRIIa expression was 8.5% ± 5% over the course of 1 month in healthy subjects. Platelet FcγRIIa expression was within 20% of the baseline value after 6 months in 71% of patients after MI. In summary, because FcγRIIa is a protein on the surface of platelets, assay conditions and antiplatelet agents do not change expression. Intra-individual variability in platelet expression of FcγRIIa is modest. Accordingly, platelet expression of FcγRIIa is a marker of increased platelet reactivity that can be reliably and repeatedly measured. Clinical Trial Registration: NCT02505217

Despite advances in interventional and pharmacologic therapy, survivors of myocardial infarction remain at an increased risk of subsequent cardiovascular events. Initial pharmacological management includes both platelet inhibition and parenteral anticoagulation, whereas long-term pharmacological therapy relies on antiplatelet therapy for prevention of thrombotic complications. Biomarkers showing ongoing thrombin generation after acute coronary syndromes suggest that anticoagulants may provide additional benefit in reducing cardiovascular events. We review the pharmacokinetics of novel anticoagulants, clinical trial results, the role of monitoring, and future directions for the use of novel oral anticoagulants in the treatment of coronary artery disease. Clinical trials have shown that long-term use of oral anticoagulants decreases the risk of cardiovascular events, but they do so at a cost of an increased risk of bleeding. Future studies will need to identify optimal treatment combinations for selected patients and conditions that address both the appropriate combination of therapy and the appropriate dosage of each agent when used in combination.

OBJECTIVE Because coronary artery bypass graft (CABG) surgery is associated with a high turnover of platelets, assessment of platelet function should enable assessment of the effect of young (RNA-containing) platelets on platelet reactivity. This study was designed to assess platelet reactivity 1 day after CABG in patients treated previously with clopidogrel or ticagrelor. METHODS Patients (n=18) with acute coronary syndrome who required urgent CABG and had been treated for up to 2 days before surgery with aspirin plus clopidogrel (n=13) or aspirin plus ticagrelor (n=5) were enrolled. Care was not altered by participation, which entailed review of medical records and taking one sample of blood 1 day after surgery. The percentage of RNA-containing platelets was quantified using thiazole orange, and platelet function was assessed by flow cytometry. RESULTS Young platelets constituted, on average, 24% of platelets (range 4-54%) and were more likely to be activated in the absence or presence of an agonist (P

A plant's ability to maintain or improve its yield under limiting conditions, such as nutrient deficiency or drought, can be strongly influenced by root system architecture (RSA), the three-dimensional distribution of the different root types in the soil. The ability to image, track and quantify these root system attributes in a dynamic fashion is a useful tool in assessing desirable genetic and physiological root traits. Recent advances in imaging technology and phenotyping software have resulted in substantive progress in describing and quantifying RSA. We have designed a hydroponic growth system which retains the three-dimensional RSA of the plant root system, while allowing for aeration, solution replenishment and the imposition of nutrient treatments, as well as high-quality imaging of the root system. The simplicity and flexibility of the system allows for modifications tailored to the RSA of different crop species and improved throughput. This paper details the recent improvements and innovations in our root growth and imaging system which allows for greater image sensitivity (detection of fine roots and other root details), higher efficiency, and a broad array of growing conditions for plants that more closely mimic those found under field conditions.

Cardiac lipomas (CL) are uncommon. Symptoms occur based on size and location. A 55 year old man presented after incidental discovery of a cardiac mass. Echocardiography (Panel A) showed a large inferoseptal mass partly obstructing the left ventricular outflow tract (LVOT) without dynamic outflow

We describe a multidisciplinary approach to forecast, rapidly detect, and characterize explosive events during the 2016–2017 eruption of Bogoslof volcano, a back-arc shallow submarine volcano in Alaska’s Aleutian arc. The eruptive sequence began in December 2016 and included about 70 discrete explosive events. Because the volcano has no local monitoring stations, we used distant stations on the nearest volcanoes, Okmok (54 km) and Makushin (72 km), combined with regional infrasound sensors and lightning detection from the Worldwide Lightning Location Network (WWLLN). Pre-eruptive seismicity was detected for 12 events during the first half of the eruption; for all other events co-eruptive signals allowed for detection only. Monitoring of activity used a combination of scheduled checks combined with automated alarms. Alarms triggered on real-time data included real-time seismic amplitude measurement (RSAM); infrasound from several arrays, the closest being on Okmok; and lightning strokes detected from WWLLN within a 20-km radius of the volcano. During periods of unrest, a multidisciplinary response team of four people fulfilled specific roles to evaluate geophysical and remote-sensing data, run event-specific ash-cloud dispersion models, ensure interagency coordination, and develop and distribute of formalized warning products. Using this approach, for events that produced ash clouds ≥7.5 km above sea level, Alaska Volcano Observatory (AVO) called emergency response partners 15 min, and issued written notices 30 min, after event onset (mean times). Factors that affect timeliness of written warnings include event size and number of data streams available; bigger events and more data both decrease uncertainty and allow for faster warnings. In remote areas where airborne ash is the primary hazard, the approach used at Bogoslof is an effective strategy for hazard mitigation.

Postopertive troponin elevation may occur without typical or atypical cardiac symptoms and is associated with an increased 30-day morbidity and mortality. The objective of the study was to implement a quality improvement initiative of postoperative troponin surveillance algorithm aimed at intensifying medical management after vascular surgery.We conducted a single-center study of postoperative troponin surveillance after vascular surgery (n = 201) at a tertiary care, academic medical center from January to December 2016. Troponin surveillance was performed on postoperative days 1-3 after carotid endarterectomy, endovascular aortic repair, infrainguinal bypass, open abdominal aortic aneurysm repair, peripheral vascular intervention, and suprainguinal bypass, regardless of cardiac symptoms. Patients with troponin I elevation (0.034 ng/mL) were managed with a treatment algorithm which included single or dual antiplatelet (AP) agent, high-intensity statin therapy, smoking cessation consultation, and outpatient cardiology consultation and stress testing. Patients with troponin elevation ≥1.0 ng/mL received inpatient cardiology consultation. We assessed adherence to the protocol for intensification of best medical therapy defined as high-dose statin therapy, increase in AP therapy, and smoking cessation consultation according to the established algorithm.Troponin elevation was recorded in 17% (34/201) of patients and was associated with cardiac symptoms in 8 patients (24%), while 26 (76%) patients had an asymptomatic abnormal troponin on postoperative surveillance. One patient was excluded due to death immediately after SUPRA, resulting in 200 patients. Troponin elevation ≥1.0 ng/mL occurred in 11 asymptomatic patients (5.5%). Any intensification of medical therapy was instituted in 76% of patients with elevated troponin and included high-intensity statin therapy (58%), increase in AP therapy (18%), and smoking cessation consultation (66%). Once an elevated troponin level was recognized, 52% of our patients received cardiology consultation with an increased likelihood (100%) in patients with troponin ≥1 ng/mL (P 0.001). Adherence to outpatient stress testing was 66%. Intensification of medical therapy was not significantly different between patients with abnormal troponin values,0.034-1.0 (n = 23) versus ≥1.0 ng/mL (n = 10); statin therapy (P = 1.0), AP (P = 0.34), and smoking cessation (P = 1.0). One-year mortality was higher in patients with postoperative troponin elevation than those with normal postoperative troponin levels (12% vs. 2.4%; P = 0.03).Routine postoperative troponin surveillance results in intensification of statin therapy in patients with asymptomatic troponin elevation. Further study is needed to determine if this approach reduces long-term cardiovascular morbidity and mortality.

RATIONALE The stable isotope ratios of water (δ2 H and δ18 O values) have been widely used to trace water in plants in a variety of physiological, ecohydrological, biogeochemical and hydrological studies. In such work, the analyte must first be extracted from samples, prior to isotopic analysis. While cryogenic vacuum distillation is currently the most widely used method reported in the literature, a variety of extraction-collection-analysis methods exist. A formal inter-method comparison on plant tissues has yet to be carried out. METHODS We performed an inter-method comparison of six plant water extraction techniques: direct vapour equilibration, microwave extraction, two unique versions of cryogenic vacuum distillation, centrifugation, and high-pressure mechanical squeezing. These methods were applied to four isotopically unique plant portions (head, stem, leaf, and root crown) of spring wheat (Triticum aestivum L.). Extracted plant water was analyzed via spectrometric (OA-ICOS) and mass-based (IRMS) analysis systems when possible. Spring wheat was grown under controlled conditions with irrigation inputs of a known isotopic composition. RESULTS The tested methods of extraction yielded markedly different isotopic signatures. Centrifugation, microwave extraction, direct vapour equilibration, and high-pressure mechanical squeezing produced water more enriched in 2 H and 18 O content. Both cryogenic vacuum distillation systems and the high-pressure mechanical squeezing method produced water more depleted in 2 H and 18 O content, depending upon the plant portion extracted. The various methods also produced differing concentrations of co-extracted organic compounds, depending on the mode of extraction. Overall, the direct vapor equilibration method outperformed all other methods. CONCLUSIONS Despite its popularity, cryogenic vacuum distillation was outperformed by the direct vapor equilibration method in terms of limited co-extraction of volatile organic compounds, rapid sample throughput, and near instantaneous returned stable isotope results. More research is now needed with other plant species, especially woody plants, to see how far the findings from this study could be extended.

Patients undergoing transcatheter aortic valve replacement (TAVR) may be at an elevated risk of platelet dysfunction, thrombocytopenia, acquired von Willebrand’s disease, and bleeding complications [(1,2)][1]. But patients undergoing TAVR may also be at high risk for platelet activation and

Dual antiplatelet therapy with aspirin and a P2Y 12 inhibitor is the treatment of choice for the prevention of atherothrombotic events in patients with acute coronary syndromes and for those undergoing percutaneous coronary interventions. The availability of different oral P2Y 12 inhibitors (clopidogrel, prasugrel, ticagrelor) has enabled physicians to contemplate switching among therapies because of specific clinical scenarios. The recent introduction of an intravenous P2Y 12 inhibitor (cangrelor) further adds to the multitude of modalities and settings in which switching therapies may occur. In clinical practice, it is not uncommon to switch P2Y 12 inhibitor, and switching may be attributed to a variety of factors. However, concerns about the safety of switching between these agents have emerged. Practice guidelines have not fully elaborated on how to switch therapies, leaving clinicians with limited guidance on when and how to switch therapies when needed. This prompted the development of this expert consensus document by key leaders from North America and Europe with expertise in basic, translational, and clinical sciences in the field of antiplatelet therapy. This expert consensus provides an overview of the pharmacology of P2Y 12 inhibitors, different modalities and definitions of switching, and available literature and recommendations for switching between P2Y 12 inhibitors.

Dual antiplatelet therapy with aspirin and a P2Y

Objective To determine whether initiation of clopidogrel before discontinuation of cangrelor would impact on the recovery of platelet reactivity. Background The active metabolite of clopidogrel cannot bind to P2Y12 when cangrelor occupies the receptor. Pharmacodynamic studies have shown that this interaction is avoided when clopidogrel is given at the end of the cangrelor infusion. We found that antiplatelet effects of another thienopyridine, prasugrel, were apparent when prasugrel was administered 0.5 hour before cangrelor was stopped. Methods Platelet function studies (light transmission aggregometry, VerifyNow, and flow cytometry) were performed on blood from patients with stable coronary artery disease who were taking aspirin when a loading dose of clopidogrel (600 mg) was given during a cangrelor infusion (0.5 and 1 hour before cangrelor was stopped). Results were compared with those obtained when clopidogrel was given immediately after cangrelor was stopped. Results Administration of clopidogrel 0.5 and 1 hour before discontinuation of the cangrelor infusion did not prevent recovery of platelet reactivity more effectively than administration at the end of the infusion. Conclusion Our results support the previously established strategy of administering clopidogrel immediately after discontinuation of cangrelor. Earlier administration increases the recovery of platelet function. (J Interven Cardiol 2015;28:415–419)

Essentials Specific reversal agents for managing severe factor Xa inhibitor-associated bleeding are lacking. We assessed 4-factor-prothrombin complex concentrate (4F-PCC) and tranexamic acid (TXA). 4F-PCC, but not TXA, reduced the prothrombin time and increased endogenous thrombin potential. These agents may be viable options for reversal of therapeutic doses of rivaroxaban. SummaryBackground Oral activated factor X inhibitors such as rivaroxaban are widely used, but specific reversal agents are lacking. Although four-factor prothrombin complex concentrate (4F-PCC) and tranexamic acid (TXA) are sometimes used to manage serious bleeding, their efficacy is unknown. Prior studies in healthy subjects taking rivaroxaban revealed that 4F-PCC partially reverses the prolonged prothrombin time (PT), and fully restores the endogenous thrombin potential (ETP). The effect of TXA has not been evaluated. Methods In this double-blind, parallel-group study, 147 healthy volunteers given rivaroxaban 20 mg twice daily for 3 days were randomized after their morning dose on day 4 to receive intravenous 4F-PCC (50 IU kg−1), TXA (1.0 g), or saline. Standardized punch biopsies were performed at baseline and after 4F-PCC, TXA or saline administration. Reversal was assessed by measuring bleeding duration and bleeding volume at biopsy sites, and by determining the PT and ETP. Results As compared with saline, 4F-PCC partially reversed the PT and completely reversed the ETP, whereas TXA had no effect. Neither 4F-PCC nor TXA reduced bleeding duration or volume. All treatments were well tolerated, with no recorded adverse events. Conclusions Although 4F-PCC reduced the PT and increased the ETP in volunteers given supratherapeutic doses of rivaroxaban, neither 4F-PCC nor TXA influenced punch biopsy bleeding.

The Joint Polar Satellite System (JPSS) will provide improved capabilities for volcano monitoring compared the previous generation of operational polar orbiting satellites. This paper outlines collaborative work between the Alaska Volcano Observatory and NOAA/NESDIS-STAR to evaluate automated objective satellite-based alerts and derived image products for volcanic cloud detection, retrieved cloud properties, and thermal emissions.

Rathayibacter toxicus is a forage grass associated Gram-positive bacterium of major concern to food safety and agriculture. This species is listed by USDA-APHIS as a plant pathogen select agent because it produces a tunicamycin-like toxin that is lethal to livestock and may be vectored by nematode species native to the U.S. The complete genomes of two strains of R. toxicus, including the type strain FH-79, were sequenced and analyzed in comparison with all available, complete R. toxicus genomes. Genome sizes ranged from 2,343,780 to 2,394,755 nucleotides, with 2079 to 2137 predicted open reading frames; all four strains showed remarkable synteny over nearly the entire genome, with only a small transposed region. A cluster of genes with similarity to the tunicamycin biosynthetic cluster from Streptomyces chartreusis was identified. The tunicamycin gene cluster (TGC) in R. toxicus contained 14 genes in two transcriptional units, with all of the functional elements for tunicamycin biosynthesis present. The TGC had a significantly lower GC content (52%) than the rest of the genome (61.5%), suggesting that the TGC may have originated from a horizontal transfer event. Further analysis indicated numerous remnants of other potential horizontal transfer events are present in the genome. In addition to the TGC, genes potentially associated with carotenoid and exopolysaccharide production, bacteriocins and secondary metabolites were identified. A CRISPR array is evident. There were relatively few plant-associated cell-wall hydrolyzing enzymes, but there were numerous secreted serine proteases that share sequence homology to the pathogenicity-associated protein Pat-1 of Clavibacter michiganensis. Overall, the genome provides clear insight into the possible mechanisms for toxin production in R. toxicus, providing a basis for future genetic approaches.

The bacterial plant pathogen Pseudomonas syringae adapts to changes in the environment by modifying its gene expression profile. In many cases, the response is mediated by the activation of extracytoplasmic function (ECF) sigma factors that direct RNA polymerase to transcribe specific sets of genes. In this study we focus on PSPTO_1043, one of ten ECF sigma factors in P. syringae pv. tomato DC3000 (DC3000). PSPTO_1043, together with PSPTO_1042, encode an RpoERsp/ChrR-like sigma/anti-sigma factor pair. Although this gene pair is unique to the P. syringae group among the pseudomonads, homologous genes can be found in photosynthetic genera such as Rhodospirillum, Thalassospira, Phaeospirillum and Parvibaculum. Using ChIP-Seq, we detected 137 putative PSPTO_1043 binding sites and identified a likely promoter motif. We characterized 13 promoter candidates, six of which regulate genes that appear to be found only in P. syringae. PSPTO_1043 responds to the presence of singlet oxygen (1O2) and tert-butyl hydroperoxide (tBOOH) and several of the genes regulated by PSPTO_1043 appear to be involved in response to oxidative stress.

We report the toxicological and pharmacokinetic properties of the synthetic, small interfering RNA (siRNA), QPI-1007, following intravitreal administration. QPI-1007 is a chemically modified siRNA designed to act via the RNA interference (RNAi) pathway to temporarily inhibit expression of the caspase 2 protein and is being developed as a neuroprotectant for the treatment of nonarteritic anterior ischemic optic neuropathy and other optic neuropathies such as glaucoma that result in the death of retinal ganglion cells. The half-life of QPI-1007 in the vitreous and retina/choroid in the Dutch Belted rabbit was about 2 days, and there was no sign of accumulation after repeated administrations at either 2- or 4-week dosing intervals in the rabbit. QPI-1007 was well tolerated in Dutch Belted rabbits following single or repeated intravitreal administrations of up to 11 doses over 9 months. Test-article-related effects were limited to the eyes, with minimal to mild vitreal cellular infiltration being the major finding, which was reversible. In repeated-dose studies, a modest reduction in B-wave amplitude obtained by electroretinography was observed in animals treated with the highest dose level tested (3 mg, which is equivalent to a 12 mg/eye human dose) that was not considered to be clinically meaningful. Administration in the rat of either a single bolus intravenous (i.v.) injection of 100 mg/kg or daily bolus i.v. injections of 75 mg/kg/day for 28 days failed to elicit any macroscopic or microscopic changes, suggesting a low risk for systemic toxicity. QPI-1007 was negative in three genetic toxicity studies. Overall, the nonclinical studies support the further development of QPI-1007.

Bacteria contain small non-coding RNAs (ncRNAs) that are typically responsible for altering transcription, translation or mRNA stability. ncRNAs are important because they often regulate virulence factors and susceptibility to various stresses. Here, the regulation of a recently described ncRNA ofPseudomonas syringaeDC3000,spot 42(now referred to asspf), was investigated. A putative RpoE binding site was identified upstream ofspfin strain DC3000. RpoE is shown to regulate the expression ofspf. Also, deletion ofspfresults in increased sensitivity to hydrogen peroxide compared with the wild-type strain, suggesting thatspfplays a role in susceptibility to oxidative stress. Furthermore, expression ofalg8is shown to be influenced byspf, suggesting that this ncRNA plays a role in alginate biosynthesis. Structural and comparative genomic analyses show this ncRNA is well conserved among the pseudomonads. The findings provide new information on the regulation and role of this ncRNA inP. syringae.

Platelets and their granular contents influence both angiogenesis and breast cancer progression. This study was performed to assess the effect of breast cancer and its treatment on platelet biology and the response to inhibition of the platelet P2Y12 receptor. Receptor-specific platelet activation and inhibition was studied for three platelet-associated proteins important in cancer angiogenesis and progression, vascular endothelial growth factor (VEGF), thrombospondin1 (TSP1), and transforming growth factor beta 1 (TGF-β1). Twenty-four women with active breast cancer and 10 healthy controls not receiving antiplatelet therapy participated in the study. Ex vivo activation of platelets in whole blood was accomplished using PAR1AP, PAR4AP, convulxin, and ADP. Platelet inhibition was accomplished using the P2Y12 receptor antagonist cangrelor (the in vitro equivalent of clopidogrel). VEGF, TSP1, and TGF-β1 were measured using standard ELISA. Platelet activation by ADP, PAR1, PAR4, and collagen receptors increased VEGF, TSP1, and TGF-β1 secretion in patients with breast cancer. Agonist-induced release of VEGF was greater in cancer patients as compared to healthy controls (p = 0.02 for ADP, p < 0.001 for PAR1AP, PAR4AP, and convulxin) despite a decrease in the efficiency of VEGF secretion in patients with breast cancer. These differences were not observed for TSP1 and TGF-β1 secretion. P2Y12 receptor inhibition decreased VEGF, TSP1, and TGF-β1 secretion. In patients with cancer, cangrelor inhibited TSP1 release to a greater extent than VEGF and TGF-β1 release. In patients with breast cancer, the magnitude of platelet inhibition achieved by cangrelor was greater than that achieved with healthy controls for all agonists and platelet proteins studied. While platelets are known to influence progression of breast cancer, our results show that breast cancer and its treatment influence the platelet phenotype by increasing the secretion of pro-angiogenic proteins following platelet activation, modulating the efficiency of platelet protein release as well as increasing the response to antiplatelet therapy.

Clinical Development Phases I-III Regulatory, Quality, Manufacturing Two recent large clinical trials that enrolled exclusively or predominantly patients with ST elevation myocardial infarction and tested direct acting anticoagulants (bivalirudin, rivaroxaban) demonstrated a reduction in mortality. While the anticoagulant properties of these agents likely contributed to the beneficial effects, this Commentary will focus on the nonanticoagulant effects and will consider the premise that pleiotropic effects may have contributed to the observed benefit. In these trials, hearts experienced ischemia/reperfusion and the myocardium was directly exposed to activated coagulation factors. Activation of proteinase-activated receptors (PARs) by thrombin and factor Xa would be predicted to increase inflammation and promote ventricular dilatation and fibrosis. Because direct acting anticoagulants effectively inhibit both free and clot-bound coagulation factors, the extent of exposure to activated coagulation factors would be expected to be reduced. The anticipated preservation of left ventricular function should contribute to a reduction in the subsequent risk of death.

Objective Thrombosis complicating the rupture of an atherosclerotic plaque can lead to arterial occlusion. Tissue factor, a membrane-bound glycoprotein, is expressed to a greater extent in atherosclerotic plaques and may be a key mediator of microthrombosis and macrothrombosis. This pilot study was designed to determine whether the angiographic presence or the extent of atherosclerosis was correlated with the activity of coagulation factors in blood. A novel computational model was used to predict whether differences in the activity of coagulation factors would alter the generation of thrombin. Methods Blood was obtained for the determination of coagulation factor activity from patients undergoing cardiac catheterization (n=50) who were grouped by the extent of their coronary artery disease (CAD). Generation of thrombin and factor (f) Xa were determined computationally. Results The activities of fII and fX were significantly lower in blood from patients with more severe CAD. Despite this, the time to clot (presumably reflecting a hypercoagulable state) was shorter in all patient groups than projected in healthy participants. Tissue factor pathway inhibitor concentrations were strongly associated with the generation of fXa and thrombin, and it is the best predictor of the time to clot. Conclusion The balance between tissue factor and tissue factor pathway inhibitor appears to be a primary determinant of a hypercoagulable state that can accompany CAD. Lower concentrations of fX and fII in blood from patients with more severe CAD, who exhibit a shorter time to clot in vitro, are consistent with the clinical observation that patients at greater risk for thrombosis can also be at greater risk for bleeding.

Despite dense cloud cover, satellite-borne commercial Synthetic Aperture Radar (SAR) enabled frequent monitoring of Merapi volcano's 2010 eruption. Near-real-time interpretation of images derived from the amplitude of the SAR signals and timely delivery of these interpretations to those responsible for warnings, allowed satellite remote sensing for the first time to play an equal role with in situ seismic, geodetic and gas monitoring in guiding life-saving decisions during a major volcanic crisis. Our remotely sensed data provide an observational chronology for the main phase of the 2010 eruption, which lasted 12 days (26 October–7 November, 2010). Unlike the prolonged low-rate and relatively low explosivity dome-forming and collapse eruptions of recent decades at Merapi, the eruption began with an explosive eruption that produced a new summit crater on 26 October and was accompanied by an ash column and pyroclastic flows that extended 8 km down the flanks. This initial explosive event was followed by smaller explosive eruptions on 29 October–1 November, then by a period of rapid dome growth on 1–4 November, which produced a summit lava dome with a volume of ~ 5 × 10 6 m 3 . A paroxysmal VEI 4 magmatic eruption (with ash column to 17 km altitude) destroyed this dome, greatly enlarged the new summit crater and produced extensive pyroclastic flows (to ~ 16 km radial distance in the Gendol drainage) and surges during the night of 4–5 November. The paroxysmal eruption was followed by a period of jetting of gas and tephra and by a second short period (12 h) of rapid dome growth on 6 November. The eruption ended with low-level ash and steam emissions that buried the 6 November dome with tephra and continued at low levels until seismicity decreased to background levels by about 23 November. Our near-real-time commercial SAR documented the explosive events on 26 October and 4–5 November and high rates of dome growth (> 25 m 3 s − 1 ). An event tree analysis for the previous 2006 Merapi eruption indicated that for lava dome extrusion rates > 1.2 m 3 s − 1 , the probability of a large (1872-scale) eruption was ~ 10%. Consequently, the order-of-magnitude greater rates in 2010, along with the explosive start of the eruption on 26 October, the large volume of lava accumulating at the summit by 4 November, and the rapid and large increases in seismic energy release, deformation and gas emissions were the basis for warnings of an unusually large eruption by the Indonesian Geological Agency's Center for Volcanology and Geologic Hazard Mitigation (CVGHM) and their Volcano Research and Technology Development Center (BPPTK) in Yogyakarta — warnings that saved thousands of lives.

The U.S. Geological Survey (USGS) deployed a transportable Doppler C-band radar during the precursory stage of the 2009 eruption of Redoubt Volcano, Alaska that provided valuable information during subsequent explosive events. We describe the capabilities of this new monitoring tool and present data captured during the Redoubt eruption. The MiniMax 250-C (MM-250C) radar detected seventeen of the nineteen largest explosive events between March 23 and April 4, 2009. Sixteen of these events reached the stratosphere (above 10 km) within 2–5 min of explosion onset. High column and proximal cloud reflectivity values (50 to 60 dBZ) were observed from many of these events, and were likely due to the formation of mm-sized accretionary tephra-ice pellets. Reflectivity data suggest that these pellets formed within the first few minutes of explosion onset. Rapid sedimentation of the mm-sized pellets was observed as a decrease in maximum detection cloud height. The volcanic cloud from the April 4 explosive event showed lower reflectivity values, due to finer particle sizes (related to dome collapse and related pyroclastic flows) and lack of significant pellet formation. Eruption durations determined by the radar were within a factor of two compared to seismic and pressure-sensor derived estimates, and were not well correlated. Ash dispersion observed by the radar was primarily in the upper troposphere below 10 km, but satellite observations indicate the presence of volcanogenic clouds in the stratosphere. This study suggests that radar is a valuable complement to traditional seismic and satellite monitoring of explosive eruptions.

Observations of volcanic lightning during the eruption of Redoubt Volcano in March and April 2009 were made with the Lightning Mapping Array. During the eruption twenty-three distinct episodes of volcanic lightning were observed. Electrical activity occurred as either a volcanic lightning storm with up to thousands of lightning discharges or as a weak electrical event with only a handful of lightning discharges. During the volcanic lightning storms we observed two phases of electrical activity: the explosive phase and the plume phase. The explosive phase consisted of very small discharges (on the order of 10–100 m) occurring directly above the vent while an explosive eruption was ongoing, whereas the plume phase was comprised of discharges occurring throughout the plume subsequent to the explosive eruption. The area of discharges during the explosive phase ranged from less than 1 km2 to 50 km2 or more. The electrical activity at the beginning of the plume phase was dominated by small discharges. Over time the horizontal extent of the flashes increased, with the largest flashes occurring at the end of the plume phase. The distribution of the horizontal size of the discharges over the lifetime of the storm indicate that the charge structure of the plume evolved from a complex and ‘clumpy’ structure to a more simple horizontally stratified structure. Plume height was shown to be a key factor in the quantity of lightning in a storm. The volcanic lightning storms occurred in plumes with column heights greater than 10 km. The tall plumes may contribute to the efficiency of charge generation through ice collisions by providing strong updrafts from the large thermal energy input from the eruption.

The 2009 eruption of Redoubt Volcano, Alaska, provided a rare opportunity to compare satellite measurements of sulfur dioxide (SO2) by the Ozone Monitoring Instrument (OMI) with airborne SO2 measurements by the Alaska Volcano Observatory (AVO). Herein we: (1) compare OMI and airborne SO2 column density values for Redoubt's tropospheric plume, (2) calculate daily SO2 masses from Mount Redoubt for the first three months of the eruption, (3) develop simple methods to convert daily measured SO2 masses into emission rates to allow satellite data to be directly integrated with the airborne SO2 emissions dataset, (4) calculate cumulative SO2 emissions from the eruption, and (5) evaluate OMI as a monitoring tool for high-latitude degassing volcanoes. A linear correlation (R2 ~ 0.75) is observed between OMI and airborne SO2 column densities. OMI daily SO2 masses for the sample period ranged from ~ 60.1 kt on 24 March to below detection limit, with an average daily SO2 mass of ~ 6.7 kt. The highest SO2 emissions were observed during the initial part of the explosive phase and the emissions exhibited an overall decreasing trend with time. OMI SO2 emission rates were derived using three methods and compared to airborne measurements. This comparison yields a linear correlation (R2 ~ 0.82) with OMI-derived emission rates consistently lower than airborne measurements. The comparison results suggest that OMI's detection limit for high latitude, springtime conditions varies from ~ 2000 to 4000 t/d. Cumulative SO2 masses calculated from daily OMI data for the sample period are estimated to range from 542 to 615 kt, with approximately half of this SO2 produced during the explosive phase of the eruption. These cumulative masses are similar in magnitude to those estimated for the 1989–90 Redoubt eruption. Strong correlations between daily OMI SO2 mass and both tephra mass and acoustic energy during the explosive phase of the eruption suggest that OMI data may be used to infer relative eruption size and explosivity. Further, when used in conjunction with complementary datasets, OMI daily SO2 masses may be used to help distinguish explosive from effusive activity and identify changes in lava extrusion rates. The results of this study suggest that OMI is a useful volcano monitoring tool to complement airborne measurements, capture explosive SO2 emissions, and provide high temporal resolution SO2 emissions data that can be used with interdisciplinary datasets to illuminate volcanic processes.

Among the events of the 2009 eruption at Redoubt Volcano, Alaska, event 5 was the best documented by radar, satellite imagery, and deposit mapping. We use the new Eulerian tephra transport model Ash3d to simulate transport and deposition of event 5 tephra at distances up to 350 km. The eruption, which started at about 1230 UTC on 23 March, 2009, sent a plume from the vent elevation (estimated at 2.3 ± 0.1 km above sea level or a.s.l.) to about 16 ± 2 km above sea level in 5 min. The plume was a few kilometers higher than would be expected for the estimated average mass eruption rate and atmospheric conditions, possibly due to release of most of the eruptive mass in the first half of the 20-minute event. The eruption injected tephra into a wind field of high shear, with weak easterly winds below ~ 3 km elevation, strong southerly winds at 6–10 km and weak westerlies above ~ 16 km. Model simulations in this wind field predicted development of a northward-migrating inverted “v”-shaped cloud with a southwest-trending arm at a few kilometers elevation, which was not visible in IR satellite images due to cloud cover, and a southeast-trending arm at > 10 km elevation that was clearly visible. Simulations also predicted a deposit distribution that strongly depended on plume height: a plume height below 15 km predicted ash deposits that were located west of those mapped, whereas good agreement was reached with a modeled plume height of 15–18 km. Field sampling of the deposit found it to contain abundant tephra aggregates, which accelerated the removal of tephra from the atmosphere. We were able to reasonably approximate the effect of aggregation on the deposit mass distribution by two methods: (1) adjusting the grain-size distribution, taking the erupted mass 250 km) locations. Modeled grain-size distributions at sample locations are also generally coarser than observed. The mismatch may result from a combination of limitations in field sampling, approximations inherent in the model, errors in the numerical wind field, and aggregation of particles larger than 0.063 mm.

© 2016 by the American Association for the Advancement of Science; all rights reserved. The March 2016 eruption of Pavlof Volcano, Alaska, produced an ash plume that caused the cancellation of more than 100 flights in North America.The eruption generated strong tremor that was recorded by seismic and remote low-frequency acoustic (infrasound) stations, including the EarthScope Transportable Array.The relationship between the tremor amplitudes and plume height changes considerably between the waxing and waning portions of the eruption. Similar hysteresis has been observed between seismic river noise and discharge during storms, suggesting that flow and erosional processes in both rivers and volcanoes can produce irreversible structural changes that are detectable in geophysical data.We propose that the time-varying relationship at Pavlof arose from changes in the tremor source related to volcanic vent erosion.This relationship may improve estimates of volcanic emissions and characterization of eruption size and intensity.

Cangrelor is an intravenous antagonist of the P2Y(12) receptor characterized by rapid, potent, predictable, and reversible platelet inhibition. However, cangrelor was not superior to clopidogrel in reducing the incidence of ischemic events in the cangrelor versus standard therapy to achieve optimal management of platelet inhibition (CHAMPION) trials. A prospectively designed platelet function substudy was performed in a selected cohort of patients to provide insight into the pharmacodynamic effects of cangrelor, particularly in regard to whether cangrelor therapy may interfere with the inhibitory effects of clopidogrel. This pre-defined substudy was conducted in a subset of patients from the CHAMPION-PCI trial (n = 230) comparing cangrelor with 600 mg of clopidogrel administered before percutaneous coronary intervention (PCI) and from the CHAMPION-PLATFORM trial (n = 4) comparing cangrelor at the time of PCI and 600 mg clopidogrel given after the PCI. Pharmacodynamic measures included P2Y12 reaction units (PRU) assessed by VerifyNow P2Y12 testing (primary endpoint marker), platelet aggregation by light transmittance aggregometry following 5 and 20 μmol/L adenosine diphosphate stimuli, and markers of platelet activation determined by flow cytometry. The primary endpoint was the percentage of patients who achieved20 % change in PRU between baseline and10 h after PCI. The main trial was stopped early limiting enrollment in the platelet substudy. A total of 167 patients had valid pharmacodynamic assessments for the primary endpoint. The percent of individuals achieving20 % change in PRU between baseline and10 h after PCI was higher with cangrelor + clopidogrel (32/84, 38.1 %) compared with placebo + clopidogrel (21/83, 25.3 %), but this was not statistically significant (difference:12.79 %, 95 % CI: -1.18 %, 26.77 %;p = 0.076). All pharmacodynamic markers as well as the prevalence of patients with high on-treatment platelet reactivity were significantly lower in patients treated with cangrelor. A rapid platelet inhibitory effect was achieved during cangrelor infusion and a rapid offset of action after treatment discontinuation. This CHAMPION platelet function substudy represents the largest pharmacodynamic experience with cangrelor, demonstrating its potent P2Y(12) receptor inhibitory effects, and rapid onset/offset of action. Although there was no significant pharmacodynamic interaction when transitioning to clopidogrel therapy, further studies are warranted given that enrollment in this study was limited due to premature interruption of the main trial.

For the treatment of patients with acute coronary syndromes in the catheterization laboratory, a high-dose bolus (HDB) regimen of tirofiban (25 µg/kg bolus, followed by an infusion of 0.15 µg/kg/min) leads to a consistent and rapid inhibition of platelet aggregation during the first hour after initiation of therapy. The objective of the present study was to use pharmacokinetic modeling to identify an appropriate dosage of tirofiban that would produce a plasma concentration-time profile in patients with severe renal impairment (creatinine clearance

The objective was to evaluate the determinants of change (Δ) in insulin sensitivity in overweight coronary artery disease male patients without diabetes after an intensive lifestyle intervention. All patients received nutritional counseling and performed 4 months of exercise training (ET) according to 1 of 2 protocols: aerobic ET (65%-70% of peak aerobic capacity [VO(2)]) 25 to 40 minutes 3 times a week (n = 30) or walking (50%-60% of peak VO(2)) 45 to 60 minutes at least 5 times a week (n = 30). Data from participants of both ET groups were pooled, and post-intensive lifestyle intervention results were compared with baseline data. The primary outcome was Δ insulin sensitivity (m-value) assessed by the criterion standard technique, the euglycemic-hyperinsulinemic clamp. Changes in weight, body mass index, total and percentage fat mass (by dual-energy x-ray absorptiometry scan), waist circumference, total abdominal and visceral fat (by computed tomographic scan), high-sensitivity C-reactive protein, peak VO(2), daily energy intake, and physical activity energy expenditure (PAEE) (by doubly labeled water technique) were also assessed. Daily energy intake decreased by 335 kcal, and PAEE increased by 482 kcal/d (all P < .0001). The mean weight loss was 6.4 kg, and the mean improvement in m-value was 1.6 mg/kg fat-free mass per minute. Univariate determinants of Δ m-value were low baseline PAEE, walking protocol, Δ weight, Δ body mass index, Δ total and percentage fat mass, Δ waist circumference, Δ total abdominal and visceral fat, and Δ PAEE (all P < .05). In multivariate analysis, the only significant determinant of Δ m-value was Δ PAEE (P < .02). In this analysis, the most powerful determinant of improved insulin sensitivity in overweight coronary artery disease patients is the change in PAEE.

Because novel direct acting anticoagulants are being tested in the secondary prevention of cardiovascular events, we assessed potential effects of a direct acting antagonist of Factor Xa on platelet function. Blood from patients with known coronary artery disease who were treated with aspirin but no other antithrombotic agent was spiked in vitro with rivaroxaban alone or in combination with a direct acting P2Y12 antagonist (cangrelor). To limit cofounding effects of anticoagulants and to enable interaction between coagulation factors, blood was anticoagulated only with a specific inhibitor of Factor XIIa, corn trypsin inhibitor. Polymerization of fibrin was prevented with the peptide GPRP. Activation of platelets was determined with the use of flow cytometry in response to lipidated tissue factor, thrombin, the collagen mimetic convulxin, and adenosine diphosphate (ADP). Rivaroxaban inhibited the activation of platelets induced by tissue factor and to a lesser extent activation induced by thrombin, effects that were accentuated when combined with cangrelor. Rivaroxaban did not attenuate convulxin-induced activation of platelets; however, a limited but consistent attenuation of ADP-induced platelet activation was seen with blood anticoagulated with rivaroxaban. Effects of rivaroxaban on ADP-induced platelet activation were not mediated by thrombin, tissue factor, or platelet-leukocyte aggregation. In conclusion, rivaroxaban attenuated in vitro the activation of platelets mediated by thrombin. In light of the pivotal role of thrombin in platelet activation after rupture of an atherosclerotic plaque, rivaroxaban should attenuate platelet activation in vivo, an effect that is accentuated by combination with a P2Y12 antagonist.

BACKGROUND: Myxoid/round cell liposarcoma (MRCL) is the second most common liposarcoma subtype, accounting for >33% of liposarcomas and approximately 10% of all soft tissue sarcomas. Although MRCL is a chemosensitive subtype, patients with metastatic disease have a poor outcome. NY-ESO-1 is a cancer-testis antigen (also known as cancer germ cell antigen) that has been successfully targeted in vaccine trials and in adoptive T-cell therapy trials for the treatment of several solid tumors. METHODS: The authors investigated the feasibility of targeting NY-ESO-1 in patients with MRCL by evaluating the prevalence of NY-ESO-1 expression in tumors using immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction analysis. NY-ESO-1–specific tumor recognition by NY-ESO-1–specific T-cells also was analyzed using a chromium release assay. RESULTS: A search of the University of Washington Sarcoma Tissue Bank identified paraffin-embedded tumor samples from 25 patients with MRCL. NY-ESO-1 expression was observed in every MRCL tumor assessed (100%); in 18 tumors (72%), staining was homogenous. In all but 2 tumors, staining was sufficiently robust (2þ) that such patients would be eligible for clinical trials of NY-ESO-1–directed therapy. By using NY-ESO-1 specific, CD8-positive T-cells, the in vitro sensitivity of myxoid liposarcoma cell lines to antigen-specific lysis was demonstrated. CONCLUSIONS: The current results establish NY-ESO-1 as an important target antigen for the treatment of patients with MRCL. Cancer 2012;118:4564-70. V C 2012 American Cancer Society.

Background More than 80% of patients entering cardiac rehabilitation are overweight, with a high prevalence of associated insulin resistance, diabetes, hypertension, hyperlipidemia, and a prothrombotic state. Because each of these characteristics is associated with abnormalities of endothelial-dependent flow-mediated dilatation (FMD), a predictor of long-term prognosis in patients with coronary heart disease (CHD), we assessed the effect of exercise training and weight reduction on FMD in overweight patients with CHD. Methods All patients (N = 38) participated in behavioral weight loss while taking their usual preventive medications. Subjects were randomized to one of two exercise protocols, which differed by caloric expenditure. The primary outcome was extent (%) of brachial artery FMD measured by ultrasonography before and after the 4-month exercise and weight-loss program. Results Both study groups experienced an increase in brachial artery FMD after weight loss and exercise. Patients randomized to the higher-caloric exercise condition (longer-distance walking) lost more weight (8.6 ± 4.1 kg vs 2.3 ± 3.3 kg [P Conclusions Exercise and weight loss increased FMD in overweight and obese patients with CHD. Greater weight reduction was associated with a greater improvement in FMD; thus, there was a dose effect. Trial registry ClinicalTrials.gov; No.: NCT00628277; URL: www.clinicaltrials.gov

Platelets are pivotal contributors to arterial thrombosis. Dual antiplatelet therapy with aspirin and clopidogrel has become the standard of care for the secondary prevention of cardiovascular events in patients with acute coronary syndromes and after percutanous coronary intervention. Clinical evidence of the continued risk of cardiovascular events plus pharmacodynamic evidence of substantial variability in on-treatment platelet reactivity has supported the development of new therapeutic strategies, using established agents and new antiplatelet drugs. This article will highlight recent pivotal clinical trials seeking to advance the use of antiplatelet therapy in patients with cardiovascular disease.

Gene expression signatures in blood correlate with specific diseases. Such signatures may serve as valuable diagnostic and prognostic tools in disease management. Blood gene expression signatures associated with heart failure may be applied to predict prognosis, monitor disease progression, and optimize treatment. Blood gene expression profiles were generated for 71 subjects with heart failure and 15 controls without heart failure, using the Affymetrix GeneChip U133Plus2.0. Survival analysis identified 197 “mortality genes” that were significantly associated with patient outcome. Functional categorization showed that genes associated with T cell receptor signaling were most significantly overpresented. Cluster analysis of these T cell receptor signaling genes significantly categorized heart failure patients into three risk groups ( P = 0.031) that were distinct from the three risk groups categorized by New York Heart Association (NYHA) Classification ( P = 0.0002). By combining the analysis of clinical assessment (NYHA class) with T cell receptor signaling gene expression, we proposed a model that demonstrated an even greater differentiation of patients at risk ( P = 0.0001). In this discovery study, we identified blood expression signatures associated with heart failure patient outcomes. Characterization of these mortality genes helped identify a set of T cell receptor signaling genes that may be of utility in predicting survival of heart failure patients. These data raise the possibility of prospectively risk stratifying patients with heart failure by integrating blood gene expression signatures with current clinical assessment.

The genus Burkholderia encompasses a fascinating collection of diverse β-proteobacteria (Coenye and Vandamme, 2003). This genus includes over 50 species, some of which are potentially useful in bioremediation, while other members are capable of forming nitrogen-fixing root nodules with legumes (Bontemps et al., 2010; Chen et al., 2003). Some members protect host plants against fungal pathogens, while others are themselves pathogenic against plants, animals, and humans (Coenye and Vandamme, 2003; Jones and Webb, 2003). Seventeen pathogenic species are members of the Burkholderia cepacia complex, or BCC (Vandamme et al., 1997; Vanlaere et al., 2008; Vanlaere et al., 2009), two of which are described by the Center for Disease Control as category B select agents (Godoy et al., 2003). B. cenocepacia, previously known as B. cepacia genomovar III (Vandamme et al., 2003), is recognized as an opportunistic pathogen of humans and is a particular threat to cystic fibrosis (CF) patients (Mahenthiralingam et al., 2005; Vandamme et al., 1997). Colonization of the CF lung by B. cenocepacia (Vandamme et al., 2003) tends to occur in patients already infected with Pseudomonas aeruginosa, another opportunistic pathogen of the CF lung (Jones and Webb, 2003; Vandamme et al., 1997). An infection caused by both organisms can result in serious clinical complications. B. cenocepacia strains are resistant to most antibiotics, making them virtually impossible to eradicate (Nzula et al., 2002). Infections with B. cenocepacia may have variable clinical outcomes ranging from asymptomatic carriage to a sudden fatal deterioration in lung function (Mahenthiralingam et al., 2005). Four strains of B. cenocepacia have been sequenced in their entirety, one of which is described in a publication (Holden et al., 2009). The Joint Genome Institute is currently sequencing nine additional strains (JGI, 2010). All four sequenced isolates have three circular chromosomes that vary in size between 3.9 and 0.88 MB in length. Strains J2315 and HI2424 also have one plasmid, 93 KB and 165 KB in length, respectively. Many or possibly all Burkholderia spp. encode at least one regulatory system that resembles the LuxR and LuxI proteins of Vibrio fischeri, where LuxI synthesizes an acylhomoserine lactone (AHL)-type pheromone, also called an autoinducer, and LuxR is an AHL-dependent transcriptional regulator (Choi and Greenberg, 1992; Eberhard et al., 1981; Engebrecht and Silverman, 1984). Regulatory systems of this family are found in countless proteobacteria, where they are thought to allow individual bacteria to coordinate their physiology with their siblings. Collectively, these systems regulate diverse processes, including pathogenesis, biofilm formation, bacterial conjugation, and the production of antibiotics and other secondary metabolites (Whitehead et al., 2001). In general, target genes are transcribed preferentially at population densities high enough to favor AHL accumulation (Eberhard et al., 1991), a phenomenon referred to as quorum sensing (Fuqua et al., 1994). B. thailandiensis has three such systems, one of which is implicated in cell aggregation, while another is required for antibiotic production (Chandler et al., 2009; Duerkop et al., 2009). A plant growth promoting isolate of B. ambifaria uses quorum sensing to regulate the production of the anti-fungal compound pyrrolnitrin (Schmidt et al., 2009). LuxR-type proteins have two domains, an N-terminal pheromone binding domain and a C-terminal DNA binding domain (Pappas et al., 2004). Purified LuxR, TraR of Agrobacterium tumefaciens, and LasR of Pseudomonas aeruginosa, when complexed with their respective AHLs, bind with high specificity to recognition sequences (referred to as lux, tra, or las boxes, respectively) that are found at target promoters (Schuster et al., 2004; Urbanowski et al., 2004; Zhu and Winans, 1999). LasR is also able to bind to sequences that have no obvious resemblance to canonical las boxes. A few members of this family bind DNA only in the absence of AHLs (Castang et al., 2006; Cui et al., 2005; Fineran et al., 2005; Minogue et al., 2005; Sjoblom et al., 2006). B. cenocepacia J2315 encodes three LuxR homologs and two LuxI homologs (Lewenza et al., 1999; Malott et al., 2005; Malott et al., 2009). Among these, CepR and CepI appear to be well conserved within the BCC (Venturi et al., 2004). CepI synthesizes primarily octanoylhomoserine lactone (OHL), and lower levels of hexanoylhomoserine lactone (HHL) (Aguilar et al., 2003b; Gotschlich et al., 2001; Huber et al., 2001; Lewenza et al., 1999). Null mutations in cepI or cepR increased the production of the siderophore ornibactin, and decreased the production of secreted lipases and metalloproteases ZmpA and ZmpB (Kooi et al., 2006; Lewenza et al., 1999; Lewenza and Sokol, 2001; Sokol et al., 2003). CepI and CepR are also required for swarming motility and biofilm formation (Huber et al., 2001) and for pathogenicity in several animal models (Kothe et al., 2003; Sokol et al., 2003). B. cenocepacia also expresses the CciI and CciR proteins, which are encoded on a genomic island called cci (cenocepacia island), that is associated with epidemic strains (Malott et al., 2005). The CepIR and CciIR systems extensively interact, in that CciR negatively regulates cepI, while CepR is required for expression of the cciIR operon (Malott et al., 2005). Transcriptional profiling studies indicate that CepR and CciR regulate many of the same genes, but do so in opposite ways (O'Grady et al., 2009). B. cenocepacia also encodes an orphan LuxR homolog called CepR2, which represses a cluster of genes that may direct production of an antibiotic or other secondary metabolite (Malott et al., 2009). In addition to transcriptional profiling several other approaches have been used to identify genes whose expression is influenced by CepR and/or OHL. In one study, the proteome of a wild type B. cenocepacia was compared to that of a cepR mutant. Fifty-five proteins were found to be differentially expressed in the two strains, approximately 10% of all detected proteins (Riedel et al., 2003). In a second study, fragments of a B. cepacia strain were cloned into a promoter trap plasmid and introduced into an E. coli strain that expressed CepR (Aguilar et al., 2003a). Twenty-eight promoter fragments were identified as being induced by OHL, and in all cases, induction required CepR. In a third study, a library of B. cenocepacia DNA fragments were introduced into a plasmid containing a promoterless luxCDABE operon (Subsin et al., 2007). That study identified 58 OHL-inducible promoters and 31 OHL-repressible promoters. Regulation of nine of these genes required CepR, while the others were not tested. Seven OHL-inducible genes were identified by screening a library of lacZ fusions (Weingart et al., 2005). Induction of all of these genes required CepR. Purified CepR-OHL complexes bound with high affinity and specificity to specific DNA sequences at two target promoters (Weingart et al., 2005). These binding sites contained a 16-nucleotide imperfect dyad symmetry and were centered approximately 44 nucleotides upstream of the transcription start sites. These two sites are to date the only experimentally confirmed CepR binding sites. Most of the studies described above do not distinguish whether a target promoter is controlled by CepR directly or indirectly. CepR could regulate a promoter indirectly, for example, by directly regulating an unknown regulatory gene whose product directly regulates that promoter. Alternatively, a CepR mutation might perturb cellular physiology in such a way that various promoters are affected by secondary effects. To date, the most comprehensive study attempting to define the optimal CepR binding site was done by Chambers, Sokol, and colleagues (Chambers et al., 2006), who approached this question with an impressive combination of genetics and bioinformatics. Mutagenesis of the known CepR binding site within the cepI promoter completely abolished induction (Chambers et al., 2006). The promoters of six genes known to be induced by OHL were used to formulate a consensus CepR binding motif (Chambers et al., 2006). This information was used to test eight additional candidate promoters, six of which were CepR-regulated. Ultimately, ten inducible promoters were used to refine the consensus sequence, and 57 possible CepR binding sites were identified upstream of various genes. The consensus motif identified in the Chambers study included the sequence CTG-N10-CAG, which has dyad symmetry. However, several other bases in the consensus did not preserve this symmetry, and some of those non-symmetric bases were said to be highly conserved (Chambers et al., 2006). The partial dyad symmetry suggests that CepR binds DNA as dimer and that the two DNA binding domains have rotational symmetry. Although we have no proof of this, structural studies of a related protein support this idea (Vannini et al., 2002; Zhang et al., 2002). Several other LuxR-type proteins are thought to decode dyad symmetrical sequences (Antunes et al., 2008; White and Winans, 2007; Whitehead et al., 2001). In the present study, we tested the ten putative CepR binding sites described above for the ability to bind purified CepR-OHL complexes. We also systematically resected and mutated a known CepR binding site, and use the resulting information to identify four new promoters that are regulated directly by CepR. All four promoters are regulated by CepR in vivo, require their binding sites for regulation, and bind with high affinity to CepR-OHL in vitro.

BACKGROUND: Nonleukoreduced units of red blood cells (RBCs) contain activated platelets (PLTs) that interact with white blood cells (WBCs) and may promote inflammation and thrombosis in the recipient. The aim of this study was to characterize PLT-WBC interactions (PLT-WBC aggregates [PLAs]), WBC apoptosis, WBC death, and the development of procoagulant activity in RBCs during storage. STUDY DESIGN AND METHODS: RBCs were prepared from volunteer donor blood and stored. Samples were analyzed with flow cytometry between Days 1 and 15 to measure PLT-monocyte aggregate (PMA) and PLT-neutrophil aggregate (PNA) formation, WBC apoptosis (annexin V binding), and cell death (binding of 7-aminoactinomycin D). Procoagulant activity in the supernatant of four RBC preparations was assessed between Days 1 and 39 using a clotting assay with and without the addition of an inhibitory anti-tissue factor (TF) antibody, αTF-5. RESULTS: PLA formation was extensive and maximal on Day 3 of storage (PNA, 23 ± 13%; PMA, 93 ± 4%; n = 6). Apoptosis was progressive throughout storage, with 95 ± 4% of neutrophils and 73 ± 19% of monocytes binding annexin V on Day 15. Cell death became measurable after apoptosis. Procoagulant activity was observed in all RBCs but with varying temporal patterns. It was partially TF dependent and removed with high-speed centrifugation, suggestive of an association with microparticles. CONCLUSION: The activation of PLTs during the storage of RBCs induces PLA formation that precedes WBC apoptosis and death. Procoagulant activity, likely associated with microparticles derived from apoptotic WBCs, may contribute to adverse effects of stored, nonleukoreduced RBCs.

Non-coding RNAs (ncRNAs) are important components of many regulatory pathways and have key roles in regulating diverse functions. In the Pseudomonads, the two-component system, GacA/S, directly regulates at least two well-characterized ncRNAs, RsmZ and RsmY, which act by sequestration of translation repressor proteins to control expression of various exoproducts. Pseudomonas fluorescens CHA0 possesses a third ncRNA, RsmX, which also participates in this regulatory pathway. In this study we confirmed expression of five rsmX ncRNAs in Pseudomonas syringae pv. tomato DC3000, and determined the distribution of the members of the rsmX ncRNA family by screening available genomic sequences of the Pseudomonads. Variable numbers of the rsmX family exist in Pseudomonas genomes, with up to five paralogs in Pseudomonas syringae strains. In Pseudomonas syringae pv. tomato DC3000, the rsmX genes are 112 to 120 nucleotides in size and are predicted by structural analysis to contain multiple exposed GGA motifs, which is consistent with structural features of the Rsm ncRNAs. We also found that these rsmX ncRNA genes share a conserved upstream region suggesting that their expression is dependent upon the global response regulator, GacA.

Molecular factors enabling microbial pathogens to cause plant diseases have been sought with increasing efficacy over three research eras that successively introduced the tools of disease physiology, single-gene molecular genetics, and genomics. From this work emerged a unified model of the interactions of biotrophic and hemibiotrophic pathogens, which posits that successful pathogens typically defeat two levels of plant defense by translocating cytoplasmic effectors that suppress the first defense (surface arrayed against microbial signatures) while evading the second defense (internally arrayed against effectors). As is predicted from this model and confirmed by sequence pattern–driven discovery of large repertoires of cytoplasmic effectors in the genomes of many pathogens, the coevolution of (hemi)biotrophic pathogens and their hosts has generated pathosystems featuring extreme complexity and apparent robustness. These findings highlight the need for a fourth research era of systems biology in which virulence factors are studied as pathosystem components, and pathosystems are studied for their emergent properties.