Evaluation of different methods for the extraction of DNA from fungal conidia by quantitative competitive PCR analysis.

Five different DNA extraction methods were evaluated for their effectiveness in recovering PCR templates from the conidia of a series of fungal species often encountered in indoor air. The test organisms were Aspergillus versicolor, Penicillium chrysogenum, Stachybotrys chartarum, Cladosporium herbarum and Alternaria alternata. The extraction methods differed in their use of different cell lysis procedures. These included grinding in liquid nitrogen, grinding at ambient temperature, sonication, glass bead milling and freeze-thawing. DNA purification and recovery from the lysates were performed using a commercially available system based on the selective binding of nucleic acids to glass milk. A simple quantitative competitive polymerase chain reaction (QC-PCR) assay was developed for use in determining copy numbers of the internal transcribed spacer (ITS) regions of the ribosomal RNA operon (rDNA) in the total DNA extracts. These quantitative analyses demonstrated that the method using glass bead milling was most effective in recovering PCR templates from each of the different types of conidia both in terms of absolute copy numbers recovered and also in terms of lowest extract to extract variability. Calculations of average template copy yield per conidium in this study indicate that the bead milling method is sufficient to support the detection of less than ten conidia of each of the different organisms in a PCR assay.

Software for real-time control of a tidal liquid ventilator.

The purpose of this project was to develop and test computer software and control algorithms designed to operate a tidal liquid ventilator. The tests were executed on a 90-MHz Pentium PC with 16 MB RAM and a prototype liquid ventilator. The software was designed using Microsoft Visual C++ (Ver. 5.0) and the Microsoft Foundation Classes. It uses a graphic user interface, is multithreaded, runs in real time, and has a built-in simulator that facilitates user education in liquid-ventilation principles. The operator can use the software to specify ventilation parameters such as the frequency of ventilation, the tidal volume, and the inspiratory-expiratory time ratio. Commands are implemented via control of the pump speed and by setting the position of two two-way solenoid-controlled valves. Data for use in monitoring and control are gathered by analog-to-digital conversion. Control strategies are implemented to maintain lung volumes and airway pressures within desired ranges, according to limits set by the operator. Also, the software allows the operator to define the shape of the flow pulse during inspiration and expiration, and to optimize perfluorochemical liquid transfer while minimizing airway pressures and maintaining the desired tidal volume. The operator can stop flow during inspiration and expiration to measure alveolar pressures. At the end of expiration, the software stores all user commands and 30 ventilation parameters into an Excel spreadsheet for later review and analysis. Use of these software and control algorithms affords user-friendly operation of a tidal liquid ventilator while providing precise control of ventilation parameters.

Identification of putative sequence specific PCR primers for detection of the toxigenic fungal species Stachybotrys chartarum.

The nucleotide sequence of a c 936 bp segment of the nuclear rRNA gene operon was determined for the toxigenic fungal species Stachybotrys chartarum and for other species of Stachybotrys and the related genus Memnoniella. This information was used to infer the phylogenetic relationships of these organisms and to search for sequence specific polymerase chain reaction (PCR) primers for S. chartarum in the internal transcribed spacer (ITS) regions. Searches for candidate primers were performed both by computer using the commercially available Oligo(R) v5.0 primer analysis software package and by manual inspection of the aligned sequences. Primers identified in both types of searches were evaluated for their specificities using a priming efficiency analysis algorithm available in the Oligo(R) 5.0 software. The automated computer searches were unsuccessful in finding S. chartarum-specific primers but did identify a group-specific reverse primer (designated as StacR4) for a phylogenetically related cluster of species that included S. chartarum. Manual searches led to the identification of a reverse primer (designated as StacR3) that was predicted to be specific for only S. chartarum and one other species of Stachybotrys. Experimental PCR analyses using these primers in conjunction with a universal forward primer indicated that the computer-generated amplification efficiency predictions were correct in most instances. A notable exception was the finding that StacR3 was specific only for S. chartarum. The relative merits of different PCR strategies for the detection of S. chartarum employing either one or both of the primers identified in this study are discussed.

Cardiopulmonary interaction during partial liquid ventilation in surfactant-treated preterm lambs.

UNLABELLED: Gas ventilation following instillation of perfluorochemical (PFC) liquid, partial liquid ventilation (PLV), improves gas exchange and pulmonary mechanics in neonatal animals and humans with severe respiratory distress. The effect of PLV on cardiac contractility, performance, pulmonary blood flow and ductal shunt has not been fully described. To this end, we evaluated these indices of cardiopulmonary function in eight conventionally gas ventilated, surfactant-treated premature lambs (125 days gestation) before and during PLV. Animals were instrumented with central venous and aortic lines. Serial evaluation of arterial blood chemistry/pressure, and pulmonary mechanics was performed; cardiac contractility, performance, pulmonary blood flow and ductal shunts were serially assessed by echocardiography. As compared to conventional gas ventilation, during PLV there was a significant decrease in left ventricular meridian (22.5+/-6.6 SE vs 8.1+/-1.4 SE g/cm2, P < 0.02) and circumferential wall stress (54.1+/-16.5 vs 24.4+/-3.8 SE g/cm2, P < 0.04) at end systole. The fall in wall stress at end systole was associated with a significant decrease in left ventricular internal diameter (1.2+/-0.05 SE vs 1.04+/-0.045 SE cm; P < 0.01). There were no significant changes in heart rate, systemic arterial and central venous pressures, systemic vascular resistance, left ventricular shortening and ejection fractions during PLV. The decrease in wall stress was associated with a significant decrease in mean airway pressures (15.9+/-1.1 SE vs 9.9+/-0.2 SE cmH2O; P < 0.05) and ostensibly a change in intrathoracic pressures during PLV. There were no significant differences in blood flows (pre vs during PLV; ml/min/kg): pulmonary (226+/-62 SE vs 293+/-65 SE), aortic (237+/-36 SE vs 204+/-21 SE), and left to right ductal (119+/-25 SE vs 105.5+/-26 SE) measured before and during PLV. CONCLUSION: Cardiac output and pulmonary blood flow do not change significantly during PLV and therefore do not appear to contribute to improved gas exchange. This stable cardiac performance occurs at lower wall stress and thereby more advantageous energetic conditions.

Effects of halothane on the immature lamb heart.

The choice of anesthesia during pregnancy and fetal operations is controversial. Halothane frequently is used, but its direct effects on fetal cardiac performance are unknown. The effects of halothane on fetal cardiac mechanics were studied in 8 fetal lamb hearts (135 days' gestation) using a modified Langendorff model connected to a membrane oxygenator. The perfusate consisted of oxygenated maternal blood at a constant flow temperature, hematocrit value, and glucose level. Coronary blood flow, left ventricular systolic pressure, left ventricular end-diastolic pressure, and the developed left ventricular pressure at a fixed volume were evaluated at baseline and after the addition of incremental concentrations of halothane to the perfusate through the oxygenator. Perfusate halothane levels were maintained in a clinical range. Systolic and diastolic cardiac function were adversely affected by the administration of even low doses of halothane, despite a concomitant increase in coronary blood flow. Because of the immaturity of their calcium transport system, fetal hearts may be particularly sensitive to the known calcium channel-blocking properties of halothane.

Chronic alterations in cardiac mechanics after fetal closed heart operation.

Pulmonary artery banding was performed in 8 fetal sheep at 125 days' gestation to evaluate the feasibility of fetal closed cardiac operation. At 135 days' gestation, the fetuses were delivered and the cardiac mechanics were evaluated using a modified Langendorff isolated heart preparation. Two of the 8 fetuses aborted spontaneously 2 and 7 days after operation. Six fetuses (75%) survived the 10 days after delivery. The left ventricular cardiac mechanics in the banded fetuses (group I) were compared with those in 8 control fetuses (group II). All fetal hearts in both groups displayed a significant volume-pressure relationship. The volume-pressure curve was found to be shifted upward and to the left in the fetal hearts after pulmonary artery banding. The left ventricles subjected to increased in utero preload secondary to pulmonary artery banding were capable of generating greater peak systolic and developed pressures. Because the fetal heart manifests a Starling type of preload reserve, as such, it demonstrates that the possibility exists for subsequent fetal development to be altered by intrauterine intervention.

High speed cine-radiographic study of aortic valve leaflet motion.

The leaflets of the aortic valve move extremely rapidly during opening and closing. To analyse this movement, radiopaque markers were placed on the aortic valves of four dogs during cardiopulmonary bypass. One to five months later the dogs were studied using x-ray, and the marker motion was recorded on cine-film at 500 frames/second. Simultaneous aortic pressure varied from 70/30 to 188/152 mmHg and heart rate from 33 to 150 bpm. Analysis of 19 systolic periods and 2500 cine-frames indicated that leaflet motion occurred in four phases: 1--rapid movement during initial opening, 2--a little movement near maximal excursion, 3--a slow movement during early closing and 4--a rapid movement to complete closure. Phases 1 and 4 averaged 17.6 and 16.5 msec respectively, and were independent of heart rate, whereas the length of phases 2 and 3 varied with heart rate. The leaflets moved 82% of their maximum excursion during rapid opening and 53% during rapid closure. Leaflet curvature reversed in both the radial and the circumferential directions during opening. The leaflet profile in the radial direction changed as follows: during rapid opening, the belly of the leaflet moved outward, first causing the leaflet to straighten and then to conform to the profile of blood flow. During closure the belly of the leaflet moved centrally, causing the leaflet to curve toward the ventricle. The leaflet profile in the circumferential direction indicated that during opening the midpoint of the leaflet leads the motion towards the aortic wall.(ABSTRACT TRUNCATED AT 250 WORDS)

Pressure-volume relationship of the fetal lamb heart.

In contrast to the adult heart, the fetal heart reportedly has little functional reserve. With increased clinical emphasis on fetal cardiac diagnosis, neonatal surgery, and the potential for future fetal cardiac intervention, it is essential that we better understand fetal cardiac function. Therefore, to demonstrate the extent of fetal cardiac preload reserve, we studied 10 fetal lambs using an isolated, isovolumic, blood-perfused heart preparation. We maintained constant afterload, inotropic state, coronary blood flow, heart rate, and perfusate blood gas values. As left ventricular (LV) volume (preload) was incrementally increased, LV end-diastolic pressure and LV peak systolic pressure were recorded. Linear regression analysis demonstrated that increases in LV developed pressures were predicted by the LV volume, demonstrating the presence of the Frank-Starling mechanism in each case. The plateau of the Starling pressure-volume curve occurred at an LV end-diastolic pressure of 12.5 +/- 4.79 mm Hg (95% confidence interval, 9.07 to 15.9 mm Hg), lower than the plateau expected in the adult heart. This implies that, in the management of fetal and immature neonatal hearts, preload reserve plays an important but limited role in cardiac reserve.

Right and left ventricular pressure-volume response to elevated pericardial pressure.

Because of the close anatomic connections, the volume in 1 ventricle can directly influence the volume in the other ventricle. We examined this ventricular mechanical coupling at elevated pericardial pressures in 6 mongrel dogs. The animals were anesthetized and were mechanically ventilated with intermittent positive-pressure ventilation. Right and left ventricular volumes and pressures and pericardial pressure were simultaneously measured during control and after infusing 25, 50, and 75 ml of saline with dextran into the pericardial cavity. The ventricular volumes were calculated from cine-radiographic positions of endocardial, radiopaque markers. In the control state, right ventricular end-diastolic volume (RVEDV) increased 9.2 +/- 0.9 ml (p less than 0.05) during expiration, whereas left ventricular end-diastolic pressure (LVEDP) increased 0.6 +/- 0.7 mmHg and left ventricular end-diastolic volume (LVEDV) decreased 0.6 +/- 0.4 ml. The increased transmural LVEDP with a decreased LVEDV indicates an apparent left ventricular distensibility decrease as right ventricular diastolic volume increased, possibly because of ventricular interdependence. At the highest pericardial pressure, RVEDV increased 6.7 +/- 1.4 ml (p less than 0.05) during expiration as LVEDP increased 1.2 +/- 0.6 mm Hg and LVEDV decreased 2.0 +/- 0.6 ml (p less than 0.05). Thus, at the higher pericardial pressures, smaller changes in RVEDV produced significantly greater changes in LVEDV. This coupling between the ventricles was further examined in 5 hearts studied postmortem. The hearts were placed in cold cardioplegic solution and balloons were inserted into both ventricles.(ABSTRACT TRUNCATED AT 250 WORDS)

Biplane ventriculography in the rat.

Biplane ventriculography was performed on nine intact anesthetized rats. Images of the left ventricle large enough for analysis were obtained by placing the rats close to the radiographic tubes (direct enlargement). Sampling rates, adequate for heart rates of 500 beats/min, were obtained by filming at 500 frames/s. From the digitized silhouettes of the left ventricle the following information was obtained (means +/- SE): end-diastolic volume 0.60 +/- 0.03 ml, end-systolic volume 0.22 +/- 0.02 ml, stroke volume 0.38 +/- 0.02 ml, ejection fraction 0.63 +/- 0.02, cardiac output 118 +/- 7 ml/min, diastolic septolateral dimension 0.41 +/- 0.01 mm, diastolic anteroposterior dimension 0.40 +/- 0.01 mm, diastolic base-to-apex dimension 1.58 +/- 0.04 mm. To determine the accuracy with which the volume of the ventricle could be measured, 11 methyl methacrylate casts of the left ventricle were made. The correlation was high (r = 0.99 +/- 0.02 ml E) between the cast volumes determined by water displacement and by use of two monoplane methods (Simpson's rule of integration and the area-length method applied to the analysis of the anteroposterior films) and a biplane method (area-length). These results demonstrate that it is possible to obtain accurate dimensions and volumes of the rat left ventricle by use of high-speed ventriculography.

Right and left ventricular pressure-volume response to respiratory maneuvers.

With respiration, right ventricular end-diastolic volume fluctuates. We examined the importance of these right ventricular volume changes on left ventricular function. In six mongrel dogs, right and left ventricular volumes and pressures and esophageal pressure were simultaneously measured during normal respiration, Valsalva maneuver, and Mueller maneuver. The right and left ventricular volumes were calculated from cineradiographic positions of endocardial radiopaque markers. Increases in right ventricular volume were associated with changes in the left ventricular (LV) pressure-volume relationship. With normal respiration, right ventricular end-diastolic volume increased 2.3 +/- 0.7 ml during inspiration, LV transmural diastolic pressure was unchanged, and LV diastolic volume decreased slightly. This effect was accentuated by the Mueller maneuver; right ventricular end-diastolic volume increased 10.4 +/- 2.3 ml (P less than 0.05), while left ventricular end-diastolic pressure increased 3.6 mmHg (P less than 0.05) without a significant change in left ventricular end-diastolic volume. Conversely, with a Valsalva maneuver, right ventricular volume decreased 6.5 +/- 1.2 ml (P less than 0.05), and left ventricular end-diastolic pressure decreased 2.2 +/- 0.5 mmHg (P less than 0.05) despite an unchanged left ventricular end-diastolic volume. These changes in the left ventricular pressure-volume relationship, secondary to changes in right ventricular volumes, are probably due to ventricular interdependence. Ventricular interdependence may also be an additional factor for the decrease in left ventricular stroke volume during inspiration.

Right and left ventricular pressure-volume response to positive end-expiratory pressure.

Cardiovascular effects of positive end-expiratory pressure (PEEP) at 20 cmH2O were examined in six mongrel dogs (11.3-15.0 kg). The dogs were anesthetized through a combination of intramuscular Innovar and gaseous anesthesia (60% N2O-40% O2). For volume measurements, radiodense tantalum screws were placed on the endocardial surface of the left and right ventricle. Esophageal and left and right ventricular pressures were measured. With the use of this preparation, the effects of positive end-expiratory pressure (PEEP = 20 cmH2O) on cardiovascular function were examined. PEEP caused right ventricular transmural pressures to decrease, 3.4 +/- 1.0 to 2.0 +/- 1.0 mmHg end-diastolic (P less than 0.05) and 29.2 +/- 2.2 to 27.9 + 2.2 mmHg peak systolic; left ventricular transmural pressures decreased, 5.9 +/- 1.6 to 1.2 +/- 1.4 mmHg end-diastolic (P less than 0.05) and 117.2 +/- 8.0 to 76.2 +/- 7.4 mmHg peak systolic (P less than 0.05). After volume loading the animal (500 ml dextran), PEEP caused similar changes in right and left ventricular pressures. Plots of end-diastolic volume versus pressure showed an increase in the apparent diastolic stiffness in both ventricles with decreased end-diastolic volume.

Cardiovascular changes from expiration to inspiration during IPPV.

In vitro studies indicated that one ventricle can influence diastolic distensibility and systolic function of the opposite ventricle. Based on these in vitro observations, ventricular interdependence was examined in 12 anesthetized dogs. Right (RV) and left (LV) ventricular volumes and pressures and esophageal pressure (EP) were simultaneously measured during intermittent positive-pressure ventilation (IPPV) at zero end-expiratory pressure. Volumes were calculated from cineradiographic positions of endocardial, radiopaque markers. A comparison of the data obtained at end expiration with the data obtained at peak inspiration shows that EP decreased (4.6 +/- 0.3 to -2.1 +/- 0.2 mmHg, P less than 0.05) and RV end-diastolic volume increased (31.0 +/- 1.3 to 36.3 +/- 1.6 ml, P less than 0.05), whereas LV end-diastolic pressure increased (6.8 +/- 1.6 to 8.3 +/- 1.5 mmHg, P less than 0.05) and LV end-diastolic volume decreased (33.6 +/- 1.3 to 32.9 +/- 1.3 ml). The data indicate decreased LV distensibility as RV diastolic volume increased. As for systolic function, a comparison of end-expiration and peak inspiration data shows that RV ejection fraction increased (0.48 +/- 0.03 to 0.55 +/- 0.03 P less than 0.05) and RV stroke volume increased 5.6 +/- 1.0 ml, whereas LV ejection fraction decreased (0.54 +/- 0.03 to 0.52 +/- 0.03, P less than 0.05) and LV stroke volume decreased 0.8 +/- 0.6 ml. The decreased LV ejection fraction and stroke volume may suggest systolic interaction between the ventricles. The data indicate that the cardiovascular response to IPPV is complex, with ventricular interdependence being an important element.

Frequency analysis approach to the origin of the first and second heart sounds.

Catheter-tipped micromanometers were used to simultaneously record left ventricular and aortic pressures, and left ventricular and aortic internal phonocardiograms in order to determine if they had a common mode of origin and propagation. Spectrographic analysis showed that even with high-pass filtration the phonocardiogram obtained with a commonly used system (Millar) contained large amounts of energy in the subaudible frequency range (below 40 Hz). It was possible to derive close facsimiles of the phonocardiograms by double differentiation of the corresponding pressure pulse and conversely to derive the pressure pulse by double integration of the phonocardiograms. The propagation velocities of the first heart sound, second heart sound, and the foot of the aortic pressure pulse were found to be similar and were respectively, 4.3 +/- 0.2, 4.6 +/- 0.3, and 4.2 +/- 0.2 m/sec (+/- SE). These data support the concept that the low frequency pressure variations produced by the heart, which predominate in the left ventricular and aortic pressure pulse waveforms, are generated and propagated in the same manner as the high frequency pressure variations, which are the first and second heart sounds.

Relationship between stroke volume and pneumocardiogram.

The intratracheal pressure oscillations produced by the heartbeat, called the pneumocardiogram, were recorded in 15 dogs. It was found that a linear relationship existed between the amplitude of the major negative deflection of the pneumocardiogram and the cardiac stroke volume, the latter determined either by indicator dilution (avg r = 0.94) or by use of an electromagnetic flow probe (avg r = 0.95). This relationship existed in animals with both an open and a closed chest. The vector of left ventricular wall motion directed toward the lungs, determined by high-speed cineradiography of the motion of a tantalum marker inserted in the wall of the left ventricle, looked very similar to the pneumocardiogram. This study showed that the pneumocardiogram can be used as a noninvasive technique to measure changes in stroke volume and is caused primarily by the motion of the cardiac walls.

Coronary arteriography in canines with calcium-enriched ioxaglate and diatrizoate.

Ioxaglate, diatrizoate, and calcium-enriched solutions of these contrast agents were compared as to their effects on left ventricular pressure, the first derivative of this pressure, left ventricular stroke volume, stroke work, and mean aortic pressure during left coronary arteriography in dogs. Ioxaglate reduced these parameters significantly less than did diatrizoate. The calcium addition ameliorated the toxic effects of both ioxaglate and diatrizoate. There was an initial reduction of stroke work during and following the contrast injections, after which increased stroke work was observed. The calcium ions did not produce further augmentation of this increased stroke work.

A computer analysis of cardiac electrograms.

Computer programs were constructed for the temporal analysis of 8 electrograms obtained from various regions of isolated dog hearts. The electrograms were recorded on photosensitive paper. A computer program written in the Fortran II language allowed an x-y digitizing table to be used to input the coordinates of each electrogram into a computer. Each electrode recording within a given cardiac cycle was digitized in this manner, and the temporal intervals between the 28 combinations of electrode pairs were calculated. The program also computed the heart rate and cardiac cycle length. Subroutine options permitted the grouping of data into experiment, record, and event numbers. All data were stored on a magnetic disk. A separate computer program written in the Fortran IV language performed basic statistical operations on each of the data files. The computer system described in the present report accurately and reliably processed over 10,000 cardiac cycles. Such a system can be used to analyze large amounts of data from sources other than electrograms. Temporal analysis of the standard electrocardiogram is offered as an example of the versatility of the programs.

Left ventricular effects on right ventricular developed pressure.

The possibility that left ventricular (LV) performance might affect right ventricular (RV) function through the myocardium was examined by using isolated, flow-perfused, paced rabbit hearts beating isovolumically. Reducing LV volume from its optimal volume to zero caused a 5.7% decrease (N = 10, P less than 0.001) in right ventricular developed pressure (RVDP). Ligating the anterior ventricular branches of the left coronary artery which in the rabbit supply the LV free wall resulted in an additional 9.3% decrease in RVDP (N = 5, P = 0.05) within 3 min of ligation. Finally, cutting the LV free wall from the atrioventricular orifice to the apex (thereby preventing any developed LV free wall force during systole) caused a 45% further decrease in RVDP (N = 2, P less than 0.02). Cineradiographic study showed that the alterations in RVDP resulting from changes in LV volume and coronary occlusion correlated significantly (N = 5, P less than 0.01) with the magnitude of septal bulging into the RV cavity during systole. The results indicate that alteration in LV free wall function and changes in LV volume can directly effect RVDP through the myocardium.