Rapid assemblage of diverse environmental fungal communities on public restroom floors.

An increasing proportion of humanity lives in urban environments where they spend most of their lives indoors. Recent molecular studies have shown that bacterial assemblages in built environments (BEs) are extremely diverse, but BE fungal diversity remains poorly understood. We applied culture-independent methods based on next-generation sequencing (NGS) of the fungal internal transcribed spacer to investigate the diversity and temporal dynamics of fungi in restrooms. Swab samples were collected weekly from three different surfaces in two public restrooms (male and female) in San Diego, CA, USA, over an 8-week period. DNA amplification and culturing methods both found that the floor samples had significantly higher fungal loads than other surfaces. NGS sequencing of floor fungal assemblages identified a total of 2550 unique phylotypes (~800 per sample), less than half of which were identifiable. Of the known fungi, the majority came from environmental sources and we found little evidence of known human skin fungi. Fungal assemblages reformed rapidly in a highly consistent manner, and the variance in the species diversity among samples was low. Overall, our study contributes to a better understanding of public restroom floor fungal communities.

Mate availability contributes to maintain the mixed-mating system in a scolytid beetle.

We investigated the mating system and population genetic structure of the beetle, Coccotrypes dactyliperda, with life history characteristics that suggest the presence of a stable mixed-mating system. We examined the genetic structure of seven populations in Israel and found significant departures from the Hardy-Weinberg equilibrium and an excess of homozygosity. Inbreeding coefficients were highly variable across populations, suggesting that low levels of outbreeding occur in nature. Experiments were conducted to determine whether the observed high inbreeding in these populations is the result of a reproductive assurance strategy. Females reared in the laboratory took longer to mate with males from the same population (inbreeding) than with males from a different population (outbreeding). These results suggest that females delayed inbreeding, and were more inclined to outbreed when possible. Thus inbreeding, which predominates in most populations, may be due to a shortage of mates for outbreeding rather than a preference for inbreeding. We conclude that C. dactyliperda has a mixed-mating system that may be maintained by a reproductive assurance strategy.

Inbreeding variability and population structure in the invasive haplodiploid palm-seed borer (Coccotrypes dactyliperda).

We investigated the mating system and population genetic structure of the invasive haplodiploid palm-seed borer Coccotrypes dactyliperda in California. We focused on whether these primarily inbreeding beetles have a 'mixed-breeding' system that includes occasional outbreeding, and whether local inbreeding coefficients (F(IS)) varied with dominant environmental factors. We also analysed the genetic structure of C. dactyliperda populations across local and regional scales. Based on the analysis of genetic variation at seven microsatellite loci in 1034 individual beetles from 59 populations, we found both high rates of inbreeding and plentiful evidence of mixed-breeding. F(IS) ranged from -0.56 to 0.90, the highest variability reported within any animal species. There was a negative correlation between F(IS) and latitude, suggesting that some latitude-associated factor affecting mating decisions influenced inbreeding rates. Multiple regressions suggested that precipitation, but not temperature, may be an important correlate. Finally, we found highly significant genetic differentiation among sites, even over short geographic distances (< 1000 m).

Angiolipoma of the esophagus: a rare clinical dilemma.

Benign tumors of the esophagus are a rare but diverse group of lesions. Although non-malignant in biology, their presence can cause significant morbidity, including dysphagia, bleeding, gastrointestinal obstruction, and even asphyxiation. Diagnosis is frequently made using radiographic and endoscopic means, even in the absence of definitive biopsy. If discovered early, endoscopic or minimally invasive techniques may be used to excise these lesions, with essentially 100% cure rates. However, if discovered late, open excision or even esophagectomy may be required. Angiolipoma represents perhaps one of the rarest of the benign entities to affect the esophagus, with only a few cases reported in the current literature. We present the case of an 85-year-old man who developed complete esophageal obstruction due to a large, pedunculated angiolipoma, requiring open surgical excision.

Molecular survey of aeroplane bacterial contamination.

AIMS: To examine bacterial contamination of passenger aircraft and to identify aeroplane environments posing the greatest potential health risk. METHODS AND RESULTS: DNA was extracted from ten environmental samples collected on four different flights (three domestic, one international) from a variety of surfaces frequently touched by passengers. PCR clone libraries were made from the DNA samples using bacterial-specific 16S ribosomal DNA (rDNA) primers. A total of 271 bacterial rDNA sequences were obtained. We used BLAST analysis of the rDNA clone sequences to identify sequences in Genbank with the highest sequence similarity. The majority of the rDNA clones obtained from aeroplane environments were more than 97% identical to rDNA sequences from cultured bacterial species. Samples collected from the cabin surfaces (e.g., tray tables and arm rests) had undetectable levels of DNA and produced no PCR products. Bacterial diversity was highest on lavatory surfaces, including door handles, toilet handles, and sink faucets. Sequence data from these surfaces detected species from 58 different bacterial genera, and many of the best BLAST hits matched rDNA sequences of cultured species known to be opportunistic pathogens. The most frequently observed species came from five genera commonly associated with humans: Streptococcus, Staphylococcus, Cornybacterium, Proprionibacterium and Kocuria. CONCLUSIONS: The results show that there is a large diversity of bacterial contamination on aeroplanes, including organisms known to be opportunistic pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results indicate that aeroplanes have the potential to spread an enormous diversity of bacterial species among passengers and destinations. Aeroplane lavatories present an especially significant concern to public health.

Cholangiocarcinoma: advocate an aggressive operative approach with adjuvant chemotherapy.

Cholangiocarcinoma presents many challenges. Prognosis is thought to be determined by conventional predictors of survival; margin status, pathologic criteria, stage, and comorbid disease. Ninety-four patients, 57 males and 37 females, underwent resections for cholangiocarcinoma between 1989 and 2000. Thirty-two patients (34%) had distal tumors, 10 had midduct lesions, and 52 had proximal/intrahepatic lesions. Thirty-four patients underwent pancreaticoduodenectomies, 23 bile duct resections alone, and 37 bile duct and concomitant hepatic resections. Tumor location did not influence mean survival (distal, 28 months +/- 23; midduct, 28 months +/- 21; and proximal, 31 months +/- 36). Operation undertaken did not alter survival (bile duct resection, 30 months +/- 37; pancreaticoduodenectomy, 27 months +/- 23; and concomitant bile duct/hepatic resection, 32 months +/- 32). TNM stage failed to predict survival: 5 stage I (29 months +/- 22), 12 stage II (41 months +/- 33), 12 stage III (33 months +/- 19), and 64 stage IV (27 months +/- 32). Tumor size did not influence survival: T1-2 (32 months +/- 33) versus T3-4 lesions (29 months +/- 25). Mean survival with negative margin (n = 67) was 34 months +/- 33, whereas microscopically positive (n = 13, 23.9 months +/- 25) or grossly positive (n = 14, 20.4 months +/- 20) margins were predictive of significantly shorter survival (P < 0.03). Adjuvant treatment (n = 41) was associated with significantly longer survival (40.5 months +/- 36) than those who received no further therapy (n = 53; 24 months +/- 24) (P = 0.05). TNM stage, tumor size, operation undertaken, and location were not associated with duration of survival after resection. Margin status was associated with duration of survival, though extended survival is possible even with positive margins. Advanced stage should not preclude aggressive resection. Without specific contraindications, an aggressive operative approach is advocated followed by adjuvant therapy.

Evaluation and refinement of tmRNA structure using gene sequences from natural microbial communities.

DNA harvested directly from complex natural microbial communities by PCR has been successfully used to predict RNase P RNA structure, and can potentially provide an abundant source of information for structural predictions of other RNAs. In this study, we utilized genetic variation in natural communities to test and refine the secondary and tertiary structural model for the bacterial tmRNA. The variability of proposed tmRNA secondary structures in different organisms and the lack of any predicted tertiary structure suggested that further refinement of the tmRNA could be useful. To increase the phylogenetic representation of tmRNA sequences, and thereby provide additional data for statistical comparative analysis, we amplified, sequenced, and compared tmRNA sequences from natural microbial communities. Using primers designed from gamma proteobacterial sequences, we determined 44 new tmRNA sequences from a variety of environmental DNA samples. Covariation analyses of these sequences, along with sequences from cultured organisms, confirmed most of the proposed tmRNA model but also provided evidence for a new tertiary interaction. This approach of gathering sequence information from natural microbial communities seems generally applicable in RNA structural analysis.

Phylogeny and host specificity of Psoroptic mange mites (Acarina: Psoroptidae) as indicated by ITS sequence data.

We used molecular phylogenetic techniques to study the systematic relationships and host specificity of Psoroptes mange mites, which are pests of numerous domestic and wild ungulates. Phylogenetic analysis of DNA sequence data from the internal transcribed spacer region 1 (ITS1) of nuclear ribosomal DNA indicated that populations of Psoroptes are not host specific. Furthermore, the currently used taxonomy of Psoroptes is not concordant with the phylogeny derived from ITS1. During the course of the study, we discovered apparent paralogous ITS sequences within individual mites as a result of varying polymerase chain reaction reaction conditions. This finding concords with other studies of ITS and suggests a cautious approach when interpreting data from ITS sequences. Host DNA contamination was also found to be a significant problem in data collection, and we report on the development of methods to overcome the problems of contamination in parasitic mites.

Phylogenetically enhanced statistical tools for RNA structure prediction.

MOTIVATION: Methods that predict the structure of molecules by looking for statistical correlation have been quite effective. Unfortunately, these methods often disregard phylogenetic information in the sequences they analyze. Here, we present a number of statistics for RNA molecular-structure prediction. Besides common pair-wise comparisons, we consider a few reasonable statistics for base-triple predictions, and present an elaborate analysis of these methods. All these statistics incorporate phylogenetic relationships of the sequences in the analysis to varying degrees, and the different nature of these tests gives a wide choice of statistical tools for RNA structure prediction. RESULTS: Starting from statistics that incorporate phylogenetic information only as independent sequence evolution models for each position of a multiple alignment, and extending this idea to a joint evolution model of two positions, we enhance the usual purely statistical methods (e.g. methods based on the Mutual Information statistic) with the use of phylogenetic information available in the sequences. In particular, we present a joint model based on the HKY evolution model, and consequently a X(2) test of independence for two positions. A significant part of this work is devoted to some mathematical analysis of these methods. We tested these statistics on regions of 16S and 23S rRNA, and tRNA.

Effects of specialization on genetic differentiation in sister species of bark beetles.

We investigated the relative importance of resource use and geography on genetic differentiation in the sister-species pair of generalist and specialist bark beetles: Dendroctonus ponderosae and D. jeffreyi (Coleoptera: Scolytidae). In two regions, where the distributions of these species overlap, we collected specimens of the generalist from multiple host species and specimens of the specialist from its single host species. Using allozyme techniques, we uncovered genetic differentiation between generalist populations on different host species in the same region (one locus in each region). However, a much stronger pattern of differentiation was found between specialist populations in the two distantly separated regions (three loci). With mtDNA, we found no significant differentiation between regions in the specialist, or among host species in the generalist, although there was some differentiation between regions in the generalist (AMOVA, P < 0.05). Overall, the generalist populations maintained approximately 10 times the genetic variation in mtDNA as the specialist populations, which suggests that the specialist either has generally smaller population sizes than the generalist, or has experienced a historical population bottleneck.

Improved statistical methods reveal direct interactions between 16S and 23S rRNA.

Recent biochemical studies have indicated a number of regions in both the 16S and 23S rRNA that are exposed on the ribosomal subunit surface. In order to predict potential interactions between these regions we applied novel phylogenetically-based statistical methods to detect correlated nucleotide changes occurring between the rRNA molecules. With these methods we discovered a number of highly significant correlated changes between different sets of nucleotides in the two ribosomal subunits. The predictions with the highest correlation values belong to regions of the rRNA subunits that are in close proximity according to recent crystal structures of the entire ribosome. We also applied a new statistical method of detecting base triple interactions within these same rRNA subunit regions. This base triple statistic predicted a number of new base triples not detected by pair-wise interaction statistics within the rRNA molecules. Our results suggest that these statistical methods may enhance the ability to detect novel structural elements both within and between RNA molecules.

Phylogenetic analyses reveal ancient duplication of estrogen receptor isoforms.

To determine the origin and evolutionary significance of a recently discovered isoform of the estrogen receptor (ERbeta), we examined the phylogenetic relationship of ERbeta to the well-known alpha isoform (ERalpha) and other steroid receptors. Our phylogenetic analyses traced the origin of ERbeta to a single duplication event at least 450 million years ago. Since this duplication, the evolution of both ER isoforms has apparently been constrained such that 80% of the amino acid positions in the DNA binding domain (DBD) and 53% of the ligand binding domain (LBD) have remained unchanged. Using the phylogenetic tree, we determined the amount of evolutionary change that had occurred in two ER isoforms. The DBD and the LBD had lower rates of evolutionary change compared to the NH(2) terminal domain. However, even with strong selective constraints on the DBD and LBD, our phylogenetic analyses demonstrate two clearly separate phylogenetic histories for ERalpha and ERbeta dating back several hundred million years. The ancient duplication of ER and the parallel evolution of the two ER isoforms suggest that, although ERalpha and ERbeta share a substantial degree of sequence identity, they play unique roles in vertebrate physiology and reproduction.

Self-injurious behavior is decreased by cyproterone acetate in adult male rhesus (Macaca mulatta).

Self-injurious behavior (SIB) presents a serious problem in laboratory macaques that cannot be socially housed for scientific reasons and among institutionalized children and adults where it is often associated with different forms of brain dysfunction. We have experienced limited success in reducing SIB in macaques by enhancing their environment with enrichment devices. Psychotropic drugs also help, but problems are associated with their use. Because sexual and aggressive behavioral problems in men have been treated with progestational drugs, we tested the efficacy of cyproterone acetate (CA, 5-10 mg/kg/week) on reducing SIB in 8 singly housed, adult male rhesus macaques. The main findings were: (1) SIB and other atypical behaviors were significantly reduced during CA treatment; (2) serum testosterone was significantly reduced during CA treatment; (3) cerebral spinal fluid (CSF) levels of 5HIAA and HVA, metabolites of serotonin and dopamine, respectively, declined significantly during CA treatment; (4) the duration of SIB positively correlated with levels of 5HIAA in CSF; but (5) sperm counts were not reduced during treatment. Thus, CA was a partially effective treatment (3 months) for adult male macaques whose behavioral problems include SIB. In summary, CA reduced SIB, overall aggression, serum testosterone, CSF 5HIAA, and CSF HVA. We hypothesized that the progestin activity of CA represses the hypothalamic gonadal axis and decreases testosterone, which in turn decreases SIB. In addition, we speculate that the decrease in 5HIAA and HVA in CSF may have been caused by progestins decreasing the activity of MAO. Therefore, the reduction of SIB may also be related to an increase in the availability of active monoamines in the CNS.

Restraining infarct expansion preserves left ventricular geometry and function after acute anteroapical infarction.

BACKGROUND: Expansion of an acute myocardial infarction predicts progressive left ventricular (LV) dilatation, functional deterioration, and early death. This study tests the hypothesis that restraining expansion of an acute infarction preserves LV geometry and resting function. METHODS AND RESULTS: In 23 sheep, snares were placed around the distal left anterior descending and second diagonal coronary arteries. In 12 sheep, infarct deformation was prevented by Marlex mesh placed over the anticipated myocardial infarct. Snared arteries were occluded 10 to 14 days later. Serial hemodynamic measurements and transdiaphragmatic quantitative echocardiograms were obtained up to 8 weeks after anteroapical infarction of 0.23 of LV mass. In sheep with mesh, circulatory hemodynamics, stroke work, and end-systolic elastance return to preinfarction values 1 week after infarction and do not change subsequently. Ventricular volumes and ejection fraction do not change after the first week postinfarction. Control animals develop large anteroapical ventricular aneurysms, increasing LV dilatation, and progressive deterioration in circulatory hemodynamics and ventricular function. At week 8, differences in LV end-diastolic pressure, cardiac output, end-diastolic and end-systolic volumes, ejection fraction, stroke work, and end-systolic elastance are significant (P<0.01) between groups. CONCLUSIONS: Preventing expansion of acute myocardial infarctions preserves LV geometry and function.

A phylogenetic approach to RNA structure prediction.

Methods based on the Mutual Information statistic (MI methods) predict structure by looking for statistical correlations between sequence positions in a set of aligned sequences. Although MI methods are often quite effective, these methods ignore the underlying phylogenetic relationships of the sequences they analyze. Thus, they cannot distinguish between correlations due to structural interactions, and spurious correlations resulting from phylogenetic history. In this paper, we introduce a method analogous to MI that incorporates phylogenetic information. We show that this method accurately recovers the structures of well-known RNA molecules. We also demonstrate, with both real and simulated data, that this phylogenetically-based method outperforms standard MI methods, and improves the ability to distinguish interacting from non-interacting positions in RNA. This method is flexible, and may be applied to the prediction of protein structure given the appropriate evolutionary model. Because this method incorporates phylogenetic data, it also has the potential to be improved with the addition of more accurate phylogenetic information, although we show that even approximate phylogenies are helpful.

Angiogenesis in transmyocardial laser revascularization. A nonspecific response to injury.

BACKGROUND: The mechanism of action of transmyocardial laser revascularization (TMR) is poorly understood. TMR has been shown to stimulate angiogenesis in porcine and canine myocardium. METHODS AND RESULTS: We examined the possibility that angiogenesis also occurs in ovine myocardium and that it is a nonspecific tissue injury response. Five Dorset sheep underwent creation of transmyocardial channels of equal diameter in both the apical and basal regions of the left ventricle through the use of both CO2 laser in 1 region and a power drill in the alternate region of the same heart. All channels were closed at 4 weeks. Histology showed channel remnants composed of granulation tissue, fibrosis, and new vessels (NV). These changes were not distinguishable on the basis of the method of channel creation. The average diameter of the channels was similar (laser, 630 +/- 180 microns; drill, 750 +/- 280 microns) (P = NS). NV with smooth muscle media were seen within the channel remnant and immediately surrounding this region using Verhoeff-Van Gieson (elastic) stain. The densities of the NV within the channel remnants were similar (laser, 1.87 +/- 1.05 NV/high-power field [hpf]; drill, 1.92 +/- 1.09 NV/hpf; P = NS), and both were significantly greater than the density of vessels in remote regions, > 5 mm from the channel center (remote laser area, 0.09 +/- 0.28 NV/hpf; remote drill area, 0.04 +/- 0.21 NV/hpf; P = NS for remote areas, P < 0.001 for laser versus remote laser, P < 0.001 for drill versus remote drill area). CONCLUSIONS: These findings demonstrate that the CO2 laser stimulates angiogenesis in normal ovine myocardium and suggest that this response represents a nonspecific reaction to tissue injury.

Transmyocardial laser revascularization fails to prevent left ventricular functional deterioration and aneurysm formation after acute myocardial infarction in sheep.

OBJECTIVE: Transmyocardial laser revascularization is an investigational technique for revascularizing ischemic myocardium in patients with inoperable coronary arterial disease. This study tests the hypothesis that laser revascularization prevents left ventricular functional deterioration and aneurysm formation after acute anteroapical myocardial infarction. METHODS: An ultrasonic ascending aortic flow probe and snares around the distal left anterior descending and second diagonal coronary arteries were placed in 26 Dorsett hybrid sheep. Ten to 14 days later, snared arteries were occluded to produce an anteroapical infarction of 23% of left ventricular mass. Before infarction 14 animals had 34 +/- 4 transmyocardial perforations in the area of the anticipated infarction made with a carbon dioxide laser. Twelve animals served as controls. Hemodynamic measurements and transdiaphragmatic quantitative echocardiograms were obtained before, immediately after, and 2, 5, and 8 weeks after infarction. Eighteen sheep completed the protocol. RESULTS: All animals had large anteroapical left ventricular aneurysms with massive ventricular enlargement. Immediately after infarction the anterior wall became thinner and dyskinetic in all sheep. At 8 weeks aneurysmal size and shape were indistinguishable between groups. Two days after infarction, laser holes were filled with fibrin. At 5 and 8 weeks the infarct consisted of dense collagen, fibroblasts, scattered calcifications, myocyte fragments, neutrophils, macrophages, and no laser holes. There were no significant differences at any time between groups for cardiac pressures or output, ventricular volumes, ejection fraction, stroke work, and the stroke work-left ventricular end-diastolic pressure index. CONCLUSION: Transmyocardial laser perforations do not revascularize acute myocardial infarction in sheep.

Papillary muscle discoordination rather than increased annular area facilitates mitral regurgitation after acute posterior myocardial infarction.

BACKGROUND: Acute posterior myocardial infarction that produces immediate mitral regurgitation alters the mitral annulus and its spatial relationship with both papillary muscles. The precise deformations that cause valve insufficiency are not understood and impair efforts to repair the valve. METHODS AND RESULTS: In six Dorsett hybrid sheep, sonomicrometry transducers were placed around the mitral annulus (6) and at the tips and bases of both papillary muscles (4). Two weeks later, three branches of the circumflex coronary artery were occluded to infarct approximately 32% of the posterior left ventricle. This infarction produced acute 2 to 3+ mitral regurgitation in all animals, as determined by color flow Doppler velocity mapping. Before and after infarction, distance measurements between sonomicrometry transducers were used to produce the three-dimensional coordinates of each transducer every 5 ms. After infarction, the area of the annulus increased only 9.2+/-6.3% at end systole (ES). In addition, the normal shortening of the posterior papillary muscle was obliterated to allow its tip to move 1.4+/-0.6 mm closer to the centroid of the annulus at ES. After infarction, the anterior papillary muscle continued to shorten normally, but at ES, its tip and base were 0.9+/-0.7 mm and 1.3+/-0.7 mm farther from the centroid, respectively. CONCLUSIONS: These deformations tend to produce a relative prolapse of leaflet tissue attached to the posterior papillary muscle and restriction of leaflet tissue attached to the anterior papillary muscle. This papillary muscle discoordination with minimal annular dilatation distorts leaflet coaptation sufficiently to produce severe mitral regurgitation.

Distortions of the mitral valve in acute ischemic mitral regurgitation.

BACKGROUND: In the absence of papillary muscle rupture, the precise deformations that cause acute postinfarction mitral valve regurgitation are not understood and impair reparative efforts. METHODS: In 6 Dorsett hybrid sheep, sonomicrometry transducers were placed around the mitral annulus (n = 6) and at the tips and bases of both papillary muscles (n = 4). Later, specific circumflex coronary arteries were occluded to infarct approximately 32% of the posterior left ventricle and produce acute 2 to 3+ mitral regurgitation. Before and after infarction, distance measurements between sonomicrometry transducers produced three-dimensional coordinates of each transducer every 5 ms. RESULTS: After infarction, the annulus dilated asymmetrically orthogonal to the line of leaflet coaptation, but the annular area increased only 9.2% +/- 6.3% (p = 0.02). At end-systole, posterior papillary muscle length increased 2.3 +/- 0.9 mm (p = 0.005); the posterior papillary muscle tip moved closer to the annular plane and centroid, and the anterior papillary muscle tip moved away. CONCLUSIONS: Small deformations in mitral valvular spatial geometry after large posterior infarctions are sufficient to produce moderate to severe mitral regurgitation. The most important changes are asymmetric annular dilatation, prolapse of leaflet tissue tethered by the posterior papillary muscle, and restriction of leaflet tissue attached to the anterior papillary muscle.

Reactivity-based coronary vasospasm independent of atherosclerosis in rhesus monkeys.

OBJECTIVES: We studied the hypothesis that in the absence of vascular pathology, coronary artery vasospasm occurs as a result of local regions of vascular muscle hyperreactivity. We aimed to explore the basis for a functional etiology of those vasospasms not explained on a structural basis. BACKGROUND: Ovariectomized rhesus monkeys (Macaca mulatta) without injury or significant vascular disease were stimulated with platelet release products, and angiograms were compared with those from vasospasms induced in human patients. METHODS: We used intracoronary (IC) injections of serotonin, thromboxane A2 (U46619), endothelin 1 or angiotensin II in concentrations 3 to 10 times that which reduced coronary artery diameter by 50%. RESULTS: Although no agent alone caused vasospasm, the combination of pathophysiologic concentrations of serotonin and the stable thromboxane A2 mimetic, U46619, injected through an IC catheter, synergistically caused coronary vasospasm on the second or third challenge in five of seven monkeys. These drug-induced vasospasms were similar to vasospasms induced by mechanical injury followed by serotonin, and to those stimulated in human IC diagnostic tests, as judged by onset, appearance, kinetics and vasodilator reversal. CONCLUSIONS: These studies in ovariectomized monkeys revealed that coronary vasospasm can be stimulated without preexisting vascular pathology, endothelial denudation or injury. Reproducible vasospasm of primate coronary arteries in response to these two endogenous pathophysiologic vasoconstrictors, which are thought to be precipitating stimuli in the etiology of vasospasm, suggests that structure-independent epicardial vasospasm can be an important element in serious cardiac ischemic events, particularly the focal, persistent vasospasms that occur without plaques or injury.