Integrative analysis of green ash phloem transcripts and proteins during an emerald ash borer infestation.

BACKGROUND: Emerald ash borer (Agrilus planipennis; EAB) is an Asian insect species that has been invasive to North America for 20 years. During this time, the emerald ash borer has killed tens of millions of American ash (Fraxinus spp) trees. Understanding the inherent defenses of susceptible American ash trees will provide information to breed new resistant varieties of ash trees. RESULTS: We have performed RNA-seq on naturally infested green ash (F. pennsylvanica) trees at low, medium and high levels of increasing EAB infestation and proteomics on low and high levels of EAB infestation. Most significant transcript changes we detected occurred between the comparison of medium and high levels of EAB infestation, indicating that the tree is not responding to EAB until it is highly infested. Our integrative analysis of the RNA-Seq and proteomics data identified 14 proteins and 4 transcripts that contribute most to the difference between highly infested and low infested trees. CONCLUSIONS: The putative functions of these transcripts and proteins suggests roles of phenylpropanoid biosynthesis and oxidation, chitinase activity, pectinesterase activity, strigolactone signaling, and protein turnover.

Mountain Pine Beetle Epidemic: An Interplay of Terpenoids in Host Defense and Insect Pheromones.

The mountain pine beetle epidemic has highlighted the complex interactions of bark beetles with conifer host defenses. In these interactions, oleoresin terpenoids and volatiles, produced and released by the host tree, can be both harmful and beneficial to the beetle's success in colonizing a tree and completing its life cycle. The insect spends almost its entire life, from egg to adult, within the bark and phloem of a pine host, exposed to large quantities of complex mixtures of oleoresin terpenoids. Conifer oleoresin comprises mostly monoterpenes and diterpene resin acids as well as many different sesquiterpenes. It functions as a major chemical and physical defense system. However, the insect has evolved host colonization behavior and enzymes for terpenoid metabolism and detoxification that allow it to overcome some of the terpenoid defenses and, importantly, to co-opt pine monoterpenes as cues for host search and as a precursor for its own pheromone system. The insect-associated microbiome also plays a role in the metabolism of conifer terpenoids.

Functions of mountain pine beetle cytochromes P450 CYP6DJ1, CYP6BW1 and CYP6BW3 in the oxidation of pine monoterpenes and diterpene resin acids.

The mountain pine beetle (MPB; Dendroctonus ponderosae) is a forest insect pest that attacks several different pine (Pinus) species in its native range of distribution in western North America. MPB are exposed for most of their life cycle to the chemical defenses of their hosts. These defenses are dominated by oleoresin secretions containing mostly various monoterpenes and diterpene resin acids (DRAs). Cytochrome P450 enzymes (P450s) of the MPB are thought to be involved in the metabolism of at least some of these defense compounds. Here we describe the cloning and characterization of three MPB P450s, CYP6DJ1, CYP6BW1 and CYP6BW3, and their functions in the oxidation of various monoterpenes and diterpene resin acids. CYP6DJ1 oxidizes the monoterpenes (+)-(4R)-limonene, (-)-(4S)-limonene and terpinolene and produces (4R,8R)-limonene-8,9-epoxide, (4R,8S)-limonene-8,9-epoxide, (4S,8S)-limonene-8,9-epoxide, (4S,8R)-limonene-8,9-epoxide, perilla alcohol and several unidentified oxidized compounds. These products of CYP6DJ1 were also identified in extracts of MPB treated with the same monoterpenes. CYP6BW1 and CYP6BW3 both oxidize the DRAs abietic acid, dehydroabietic acid, neoabietic acid, levopimaric acid, palustric acid, and isopimaric acid, producing hydroxylated and epoxidized DRAs. CYP6DJ1, CYP6BW1 and CYP6BW3 appear to contribute to the metabolism of oleoresin terpenes as part of the MPB's ability to cope with host defenses.

The cytochrome P450 CYP6DE1 catalyzes the conversion of α-pinene into the mountain pine beetle aggregation pheromone trans-verbenol.

The recent outbreak of the mountain pine beetle (Dendroctonus ponderosae; MPB) has affected over 20 M hectares of pine forests in western North America. During the colonization of host trees, female MPB release the aggregation pheromone (-)-trans-verbenol. (-)-trans-Verbenol is thought to be produced from the pine defense compound (-)-α-pinene by cytochrome P450 (P450) dependent hydroxylation. MPB may also use P450s for the detoxification of other monoterpenes of the pine defense system. Here we describe the functional characterization of MPB CYP6DE1. CYP6DE1, but not the closely related CYP6DE2, used the bicyclic monoterpenes (-)-α-pinene, (+)-α-pinene, (-)-ß-pinene, (+)-ß-pinene and (+)-3-carene as substrates. CYP6DE1 was not active with other monoterpenes or diterpene resin acids that were tested as substrates. trans-Verbenol is the major product of CYP6DE1 activity with (-)-α-pinene or (+)-α-pinene as substrates. When tested with blends of different ratios of (-)-α-pinene and (+)-α-pinene, CYP6DE1 produced trans-verbenol with an enantiomeric profile that was similar to that produced by female MPB exposed to the α-pinene enantiomers.

Cytochromes P450 Preferentially Expressed in Antennae of the Mountain Pine Beetle.

The mountain pine beetle (MPB, Dendroctonus ponderosae) is a forest insect pest endemic to western North America. During dispersal and host colonization, MPB identify suitable host trees by olfaction of monoterpene volatiles, contend with host terpene defenses, and communicate with conspecifics using terpenoid and other pheromones. Cytochromes P450 (P450s) have been proposed to function in MPB olfaction, terpene detoxification, and pheromone biosynthesis. Here, we identified P450s that were abundant in the antennae transcriptome. Analysis of transcript levels across different life stages and tissues in females and males showed additional expression of most of these P450s in the midgut or fat body. These expression profiles suggest specific or overlapping functions in olfaction, detoxification, and pheromone biosynthesis.

Monoterpenyl esters in juvenile mountain pine beetle and sex-specific release of the aggregation pheromone trans-verbenol.

A recent outbreak of mountain pine beetle (MPB) has spread over more than 25 million hectares of pine forests in western North America, affecting pine species of sensitive boreal and mountain ecosystems. During initial host colonization, female MPB produce and release the aggregation pheromone trans-verbenol to coordinate a mass attack of individual trees. trans-Verbenol is formed by hydroxylation of α-pinene, a monoterpene of the pine oleoresin defense. It is thought that adult females produce and immediately release trans-verbenol when encountering α-pinene on a new host tree. Here, we show that both sexes of MPB accumulate the monoterpenyl esters verbenyl oleate and verbenyl palmitate during their development in the brood tree. Verbenyl oleate and verbenyl palmitate were retained in adult female MPB until the time of emergence from brood trees, but were depleted in males. Adult females released trans-verbenol in response to treatment with juvenile hormone III (JHIII). While both sexes produced verbenyl esters when exposed to α-pinene, only females responded to JHIII with release of trans-verbenol. Accumulation of verbenyl esters at earlier life stages may allow adult females to release the aggregation pheromone trans-verbenol upon landing on a new host tree, independent of access to α-pinene. Formation of verbenyl esters may be part of a general detoxification system to overcome host monoterpene defenses in both sexes, from which a specialized and female-specific system of pheromone biosynthesis and release may have evolved.

Toxicity of Pine Monoterpenes to Mountain Pine Beetle.

The mountain pine beetle (Dendroctonus ponderosae; MPB) is an eruptive bark beetle species affecting pine forests of western North America. MPB are exposed to volatile monoterpenes, which are important host defense chemicals. We assessed the toxicity of the ten most abundant monoterpenes of lodgepole pine (Pinus contorta), a major host in the current MPB epidemic, against adult MPB from two locations in British Columbia, Canada. Monoterpenes were tested as individual volatiles and included (-)-ß-phellandrene, (+)-3-carene, myrcene, terpinolene, and both enantiomers of α-pinene, ß-pinene and limonene. Dose-mortality experiments identified (-)-limonene as the most toxic (LC50: 32 µL/L), and (-)-α-pinene (LC50: 290 µL/L) and terpinolene (LC50: >500 µL/L) as the least toxic. MPB body weight had a significant positive effect on the ability to survive most monoterpene volatiles, while sex did not have a significant effect with most monoterpenes. This study helps to quantitatively define the effects of individual monoterpenes towards MPB mortality, which is critical when assessing the variable monoterpene chemical defense profiles of its host species.

Frontalin pheromone biosynthesis in the mountain pine beetle, Dendroctonus ponderosae, and the role of isoprenyl diphosphate synthases.

The mountain pine beetle (Dendroctonus ponderosae Hopkins) is the most destructive pest of western North American pine forests. Adult males produce frontalin, an eight-carbon antiaggregation pheromone, via the mevalonate pathway, as part of several pheromones that initiate and modulate the mass attack of host trees. Frontalin acts as a pheromone, attractant, or kairomone in most Dendroctonus species, other insects, and even elephants. 6-Methylhept-6-en-2-one, a frontalin precursor, is hypothesized to originate from 10-carbon geranyl diphosphate (GPP), 15-carbon farnesyl diphosphate (FPP), or 20-carbon geranylgeranyl diphosphate (GGPP) via a dioxygenase- or cytochrome P450-mediated carbon-carbon bond cleavage. To investigate the role of isoprenyl diphosphate synthases in pheromone biosynthesis, we characterized a bifunctional GPP/FPP synthase and a GGPP synthase in the mountain pine beetle. The ratio of GPP to FPP produced by the GPP/FPP synthase was highly dependent on the ratio of the substrates isopentenyl diphosphate and dimethylallyl diphosphate used in the assay. Transcript levels in various tissues and life stages suggested that GGPP rather than GPP or FPP is used as a precursor to frontalin. Reduction of transcript levels by RNA interference of the isoprenyl diphosphate synthases identified GGPP synthase as having the largest effect on frontalin production, suggesting that frontalin is derived from a 20-carbon isoprenoid precursor rather than from the 10- or 15-carbon precursors.

The effect of NavX on fluoroscopy times in pediatric catheter ablation.

PURPOSE: Catheter ablation is the established curative therapy for pediatric tachyarrhythmias. However, exposure to ionizing radiation from fluoroscopy during the procedure is of concern to both patients and caregivers. We sought to assess the impact of an impedance-based three-dimensional navigation system (NavX(TM), Endocardial Solutions, Inc., St. Paul, MN) on pediatric catheter ablation procedures. METHODS: We retrospectively analyzed procedural data during a 7-year period (2002-2008), which spanned the transition between standard fluoroscopic mapping and adoption of NavX(TM) mapping for catheter ablation of atrioventricular nodal reentrant tachycardia (AVNRT) and right/left-sided accessory pathways (RAP/LAP). Comparisons of total procedure time, total fluoroscopy time, and ablation fluoroscopy time (from insertion of ablation catheter until completion of procedure) between NavX(TM) and conventional mapping were made. RESULTS: Three hundred eighty-eight patients (aged 1-18 years, M/F 236:183) underwent ablation of AVNRT (n = 101), LAP (n = 130), or RAP (n = 157) using either conventional (n = 70) or NavX(TM) (n = 318) mapping. Overall success rates were similar between the two mapping approaches (95.7% for conventional versus 95.9% for NavX(TM)). NavX(TM) mapping significantly reduced ablation fluoroscopy time (15.9 ± 14.3 versus 11.0 ± 8.9 min for NavX(TM), p < 0.01) with a trend towards a decrease in total fluoroscopy time (26.4 ± 15.6 versus 23.8 ± 11.1 min for NavX(TM), p = 0.095). Total procedure time was not significantly different between the two methods (210.1 ± 66 versus 222.8 ± 61 min for NavX(TM), p = 0.13). When analyzed by arrhythmia substrate, there were significant reductions in ablation fluoroscopy time for both LAP and RAP. CONCLUSIONS: NavX(TM) mapping reduced ablation fluoroscopy times for accessory pathways during pediatric catheter ablation.

Are implantable loop recorders useful in detecting arrhythmias in children with unexplained syncope?

INTRODUCTION: Syncope and presyncope are symptoms that occur infrequently in children, are unpredictable, and represent a diagnostic challenge to the physician. Conventional diagnostic investigations are often unable to establish a diagnosis, making it difficult to determine patient risk and direct appropriate therapy. The implantable loop recorder (ILR) is a medical device that was created for prolonged monitoring of heart rate and rhythm and has been used in a limited number of pediatric studies in which the cause of the syncope is unknown. METHODS: This is a retrospective review of the clinical, surgical, and follow-up data of patients who had ILR devices implanted after conventional testing failed to identify a cause for their symptoms. RESULTS: The diagnostic yield of the ILR device in unmasking the cause for symptoms in our patient cohort was 64%. In our study, manually activated events accounted for 71% of all documented episodes and 68% of the cases involving hemodynamically important arrhythmias or transient rhythm changes. The ILR device can be safely implanted and explanted in children without significant morbidity, in most cases. None of our patients experienced any long-term adverse events associated with placement of the device and all were alive at last follow-up. CONCLUSIONS: The use of the ILR device is a useful tool to help unmask arrhythmias as a cause of unexplained syncope in children. Patient selection for who should and should not have an ILR device implanted will continue to influence its diagnostic utility and generate controversy among stakeholders.

A prospective pediatric clinical trial of digital music players: do they interfere with pacemakers?

BACKGROUND: There has been recent concern in the media about iPod interference with pacemakers. We systematically tested two types of digital music players (DMP) on pacemaker or ICD function in children. METHODS/RESULTS: Patients were monitored by a 6-lead ECG and programmer telemetry. The pacemaker was tested in bipolar and unipolar sensing at normal and maximal sensitivities. The order of DMP tested was randomized. Each DMP was placed on top of the pacemaker pocket, put into "play" mode three times (5 seconds each), with and without programmer wand placed one cm adjacent to DMP. Pacemaker interference was defined as: pacing inhibition, inappropriate pacing, oversensing, or detection of high-rate episodes associated with the use of DMP. Of the 67 patients (mean age 12 +/- 5 years), 62 had pacemakers and five had ICDs; 39 endocardial and 28 epicardial systems. Patients were tested with Sansa (67), iPod 30 GB (51), and other DMPs (43). There was no evidence of interference with pacemaker function by any DMP under any of the study conditions (each performed in triplicate). Reproducible programmer telemetry interference was shown in 11 cases (Medtronic 6/47, St. Jude Medical 5/18, Guidant 0/2) related to use of iPod (6), Sansa (1), or others (4). None of these were associated with any evidence of pacemaker malfunction. CONCLUSION: Interference with pacemaker function could not be shown with iPod or MP3 players. No additional precaution beyond standard is necessary for patients with pacemakers when they are using these players.

Evaluation of computerized interpretation of the pediatric electrocardiogram.

The purpose of this research was to determine the frequency and factors affecting disagreement between pediatric cardiologist (MD1 or MD2) and the computer-assisted interpretation (CAI) of pediatric electrocardiograms from patients with heart disease (HD, n = 586) or normal heart (n = 561). Significant disagreement was found in HD (146/586, 25%) compared with normal heart (64/561, 11%) (P < .001). The CAI overinterpreted prolonged QT, sinus rhythm with ectopy, and right ventricular hypertrophy; CAI underinterpreted sinus rhythm, sinus arrhythmia, and right bundle branch block (P < .05). Increased disagreement was independently associated with HD (odds ration [OR], 2.2), younger patient age at the time of the electrocardiogram, if the computer interpretation had more than 3 separate diagnostic statements (OR, 3.2) and if the overreading cardiologist was MD1 (OR, 2.9). Although CAI is helpful, pediatric cardiologists were more likely to disagree with the computer in rhythm diagnosis, recognition of bundle branch block, hypertrophy, and QT interval analysis.

Chronotropic incompetence in young patients with late postoperative atrial flutter: a case-control study.

AIMS: Atrial flutter causes late postoperative morbidity in congenital heart disease (CHD). Sinoatrial node dysfunction is associated with late postoperative atrial flutter, but pacing interventions driven by minimum heart rates (HR) have yielded mixed results. METHODS AND RESULTS: A retrospective case-control study was used to test the hypothesis that late postoperative atrial flutter is associated with chronotropic incompetence in active young CHD patients. Control CHD patients aged < or =18 years without documented supraventricular ectopy (n = 42) were matched with 42 patients (cases) having atrial flutter onset > or =6 months postoperatively. Minimum, average, and maximum non-flutter HRs were obtained from outpatient ambulatory 24 h ECG (Holter) recordings and graded exercise tests. Chronotropic competence was assessed using percentage of age-specific predicted maximum HR achieved, and calculated chronotropic index. Effects of rate-adaptive programming and maximum atrial pacing rates were analysed in 19 permanently paced cases. Least square estimates of minimum HRs were similar in cases and controls (54+/-2 vs. 52+/-2 bpm). Average HRs were lower in cases (75+/-2 vs. 81+/-2 bpm, P=0.02). Cases and controls differed most significantly with respect to percentage of predicted maximum HR achieved (67+/-2 vs. 80+/-2%, P < 0.001). This difference remained highly significant when the data were adjusted for age, sex, permanent pacing, and negatively chronotropic medication usage at the time of testing. Among paced patients, atrial flutter was significantly less likely to be observed in the setting of rate-adaptive pacing [odds ratio (OR) = 0.36; P < 0.05], and the likelihood of detecting atrial flutter decreased relative to the maximum programmed atrial pacing rate (OR 0.87 for every 5% increment in maximum pacing rate relative to maximum predicted HR for age; P < 0.05). CONCLUSION: Late postoperative atrial flutter is associated with chronotropic incompetence in paediatric CHD patients.

Natural history of postoperative heart block in congenital heart disease: implications for pacing intervention.

After half a century of major progress in congenital heart disease management, atrioventricular conduction block continues to complicate 1-3% of surgical procedures. Unless treated with an implanted pacemaker, permanent postoperative heart block is associated with 28-100% mortality. Postoperative heart block often proves to be transient, typically resolving within 10 days of onset. The duration of postoperative heart block is widely used as a key determinant for permanent pacemaker implantation. Current professional pacemaker implantation guidelines are largely based on this criterion. However, available natural history data suggest that other factors, such as residual conduction system injury, likely play a role in increasingly recognized cases of very late postoperative mortality and morbidity in patients who have experienced transient postoperative heart block. As growing numbers of congenital heart disease patients survive into adulthood, and artificial pacemaking capabilities continue to improve, it might be necessary to reconsider and refine currently accepted pacing indications for postoperative heart block.

Usability assessment of pacemaker programmers.

There is a perception among clinicians of usability differences in the user interface of pacemaker programmers, but there is an absence of literature in this area. The purpose of this study was to describe usability differences in pacemaker programmers. Forty-two programmer users completed self-administered questionnaires and two usability experts independently performed heuristic evaluation to identify features that violated general usability principles. Programmers from seven manufacturers (coded A-G) were evaluated. There was a balanced representation of users: nurses (58%) versus technologists (40%) who are employed in community (50%) versus academic (45%) hospitals, novice versus expert users based on the median users' programming experience of 60 months (range 1-300 months). Significant differences between programmers were found in overall user satisfaction and ease of programmer use (P < 0.0001) in the display, controls, operation, and physical dimension of the programmers (P < 0.05). Heuristic evaluations showed frequent violations of usability principles in all programmers. Problematic areas include reliance on user recall, inconsistency in operation of critical controls, poor readability, and not anticipating user wants or action. Programmer interface designs do not consistently meet user needs or general usability principles. This impacts on the safe and effective use of programmers. Guidelines in programmer design should be established, particularly with respect to labeling, location, and operation of critical controls.