CryoGrid-PIXUL-RNA: high throughput RNA isolation platform for tissue transcript analysis.

BACKGROUND: Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in tissue multiomics analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose 96-well microplate sonicator, PIXUL, that can be used in multiple workflows to extract analytes from cultured cells and tissue fragments for various downstream molecular assays. And yet, the sample preparation devices, such as PIXUL, along with the downstream multiomics analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such biospecimens remain, in comparison, inefficient. RESULTS: To mitigate this tissue interrogation bottleneck, we have developed a low-cost user-friendly system, CryoGrid, to catalog, cryostore and sample tissue fragments. TRIzol is widely used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. Columns are also commonly used to extract RNA but they involve many steps, are prone to human errors, and are also expensive. Both TRIzol and column protocols use test tubes. We developed a microplate PIXUL-based TRIzol-free and column-free RNA isolation protocol that uses a buffer containing proteinase K (PK buffer). We have integrated the CryoGrid system with PIXUL-based PK buffer, TRIzol, and PureLink column methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. CryoGrid-PIXUL, when integrated with either PK buffer, TRIzol or PureLink column RNA isolation protocols, yielded similar transcript profiles in frozen organs (brain, heart, kidney and liver) from a mouse model of sepsis. CONCLUSIONS: RNA isolation using the CryoGrid-PIXUL system combined with the 96-well microplate PK buffer method offers an inexpensive user-friendly high throughput workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions.

PIXUL-ChIP: integrated high-throughput sample preparation and analytical platform for epigenetic studies.

Chromatin immunoprecipitation (ChIP) is the most widely used approach for identification of genome-associated proteins and their modifications. We have previously introduced a microplate-based ChIP platform, Matrix ChIP, where the entire ChIP procedure is done on the same plate without sample transfers. Compared to conventional ChIP protocols, the Matrix ChIP assay is faster and has increased throughput. However, even with microplate ChIP assays, sample preparation and chromatin fragmentation (which is required to map genomic locations) remains a major bottleneck. We have developed a novel technology (termed 'PIXUL') utilizing an array of ultrasound transducers for simultaneous shearing of samples in standard 96-well microplates. We integrated PIXUL with Matrix ChIP ('PIXUL-ChIP'), that allows for fast, reproducible, low-cost and high-throughput sample preparation and ChIP analysis of 96 samples (cell culture or tissues) in one day. Further, we demonstrated that chromatin prepared using PIXUL can be used in an existing ChIP-seq workflow. Thus, the high-throughput capacity of PIXUL-ChIP provides the means to carry out ChIP-qPCR or ChIP-seq experiments involving dozens of samples. Given the complexity of epigenetic processes, the use of PIXUL-ChIP will advance our understanding of these processes in health and disease, as well as facilitate screening of epigenetic drugs.

Ultrasound-based cell sorting with microbubbles: A feasibility study.

The isolation and sorting of cells is an important process in research and hospital labs. Most large research and commercial labs incorporate fluorescently or magnetically labeled antibodies adherent to cell surface antigens for cell identification and separation. In this paper, a process is described that merges biochemical labeling with ultrasound-based separation. Instead of lasers and fluorophore tags, or magnets and magnetic particle tags, the technique uses ultrasound and microbubble tags. Streptavidin-labeled microbubbles were mixed with a human acute lymphoblastic leukemia cell line, CCL 119, conjugated with biotinylated anti-CD7 antibodies. Tagged cells were forced under ultrasound, and their displacement and velocity quantified. Differential displacement in a flow stream was quantified against erythrocytes, which showed almost no displacement under ultrasound. A model for the acoustic radiation force on the conjugated pairs compares favorably with observations. This technology may improve on current time-consuming and costly purification procedures.

Size of Sclerosing Foams Prepared by Ultrasound, Mechanical Agitation, and the Handmade Tessari Method for Treatment of Varicose Veins.

OBJECTIVES: Sclerotherapy is a therapeutic method used in the treatment of varicose veins and works by occluding damaged blood vessels with a chemical solution. Foam sclerotherapy is an attractive treatment because the results are more effective than those obtained by using liquid sclerosants. However, serious neurologic complications, which are likely related to air embolism, have been reported after treatment with foams generated by the handmade method (Tessari technique) most often used clinically. We present an alternative ultrasonic technique for preparation of sclerosing foams to treat varicose veins. METHODS: Three methods of foam generation were compared: ultrasound, mechanical agitation, and Tessari techniques. RESULTS: Optical microscopic analyses showed that low-frequency ultrasound can generate foams with smaller bubble distributions compared to those produced by handmade and mechanical agitation methods: 98% of the bubble population was less than 55 ± 10 µm for sonicated foams (mean ± SD, 19 ± 1.8 µm; maximum bubble size, <138.3 ± 32.5 µm), 196.7 ± 38.2 µm for mechanically agitated foams (mean, 37.1 ± 10.6 µm; maximum bubble size, <350 ± 70.9 µm), and 211.7 ± 20.8 µm for handmade foams (mean, 30.8 ± 3.8 µm; maximum bubble size, <445 ± 32.8 µm). CONCLUSIONS: Low-frequency ultrasonic foam generation yields smaller bubbles and more uniform size distributions than other investigated methods. These properties may reduce serious adverse events reported for sclerotherapy of varicose veins, increasing the safety of foam treatment.

Acoustic field characterization of the Duolith: measurements and modeling of a clinical shock wave therapy device.

Extracorporeal shock wave therapy (ESWT) uses acoustic pulses to treat certain musculoskeletal disorders. In this paper the acoustic field of a clinical portable ESWT device (Duolith SD1) was characterized. Field mapping was performed in water for two different standoffs of the electromagnetic head (15 or 30 mm) using a fiber optic probe hydrophone. Peak positive pressures at the focus ranged from 2 to 45 MPa, while peak negative pressures ranged from -2 to -11 MPa. Pulse rise times ranged from 8 to 500 ns; shock formation did not occur for any machine settings. The maximum standard deviation in peak pressure at the focus was 1.2%, indicating that the Duolith SD1 generates stable pulses. The results compare qualitatively, but not quantitatively with manufacturer specifications. Simulations were carried out for the short standoff by matching a Khokhlov-Zabolotskaya-Kuznetzov equation to the measured field at a plane near the source, and then propagating the wave outward. The results of modeling agree well with experimental data. The model was used to analyze the spatial structure of the peak pressures. Predictions from the model suggest that a true shock wave could be obtained in water if the initial pressure output of the device were doubled.

Comparative Study of Histotripsy Pulse Parameters Used to Inactivate Escherichia coli in Suspension.

OBJECTIVE: Bacterial loads can be effectively reduced using cavitation-mediated focused ultrasound, or histotripsy. In this study, gram-negative bacteria (Escherichia coli) in suspension were used as model bacteria to evaluate the effectiveness of two regimens of histotripsy treatments: cavitation histotripsy (CH) and boiling histotripsy (BH). METHODS: Ten-milliliter volumes of Escherichia coli were treated at different negative focal pressure amplitudes and over time periods up to 40 min. Cavitation activity was characterized with coaxial passive cavitation detection (PCD) and synchronized plane wave B-mode imaging. RESULTS: CH treatments exhibited a threshold behavior that was consistent with PCD metrics of cavitation. Above the threshold, bacterial inactivation followed a monotonically increasing log-linear relationship that indicated an exponential inactivation rate. BH exhibited no threshold, but instead followed a different monotonically increasing inactivation rate. Inactivation rates were larger for BH at or below the CH threshold, and larger for CH substantially above the threshold. CH studies performed at different pulse lengths at the same duty cycle had similar inactivation rates, suggesting that at any given pressure amplitude, the "on time" was the most important variable for inactivating E. coli. The maximum inactivation was produced by CH at the highest pressure amplitudes used, leading to a log reduction >4.2 for a 40 min treatment. CONCLUSION: The results of this study suggest that both CH and BH can be used to inactivate E. coli in suspension, with the optimal regimen depending on the attainable peak negative focal pressure at the target.

Blood vessel deformations on microsecond time scales by ultrasonic cavitation.

Transient interactions among ultrasound, microbubbles, and microvessels were studied using high-speed photomicrography. We observed liquid jets, vessel distention (motion outward against the surrounding tissue), and vessel invagination (motion inward toward the lumen). Contrary to current paradigms, liquid jets were directed away from the nearest vessel wall and invagination exceeded distention. These observations provide insight into the mechanics of bubble-vessel interactions, which appear to depend qualitatively upon the mechanical properties of biological tissues.

Sonographic Features of Abscess Maturation in a Porcine Model.

Abscesses are walled-off collections of infected fluids that often develop as complications in the setting of surgery and trauma. Treatment is usually limited to percutaneous catheterization with a course of antibiotics. As an alternative to current treatment strategies, a histotripsy approach was developed and tested in a novel porcine animal model. The goal of this article is to use advanced ultrasound imaging modes to extract sonographic features associated with the progression of abscess development in a porcine model. Intramuscular or subcutaneous injections of a bi-microbial bacteria mixture plus dextran particles as an irritant led to identifiable abscesses over a 2 to 3 wk period. Selected abscesses were imaged at least weekly with B-mode, 3-D B-mode, shear-wave elastography and plane-wave Doppler imaging. Mature abscesses were characterized by a well-defined core of varying echogenicity surrounded by a hypoechoic capsule that was highly vascularized on Doppler imaging. 3-D imaging demonstrated the natural history of abscess morphology, with the abscess becoming less complex in shape and increasing in volume. Furthermore, shear-wave elastography demonstrated variations in stiffness as phlegmon becomes abscess and then liquefies, over time. These ultrasound features potentially provide biomarkers to aid in selection of treatment strategies for abscesses.

Treating Porcine Abscesses with Histotripsy: A Pilot Study.

Infected abscesses are walled-off collections of pus and bacteria. They are a common sequela of complications in the setting of surgery, trauma, systemic infections and other disease states. Current treatment is typically limited to antibiotics with long-term catheter drainage, or surgical washout when inaccessible to percutaneous drainage or unresponsive to initial care efforts. Antibiotic resistance is also a growing concern. Although bacteria can develop drug resistance, they remain susceptible to thermal and mechanical damage. In particular, short pulses of focused ultrasound (i.e., histotripsy) generate mechanical damage through localized cavitation, representing a potential new paradigm for treating abscesses non-invasively, without the need for long-term catheterization and antibiotics. In this pilot study, boiling and cavitation histotripsy treatments were applied to subcutaneous and intramuscular abscesses developed in a novel porcine model. Ultrasound imaging was used to evaluate abscess maturity for treatment monitoring and assessment of post-treatment outcomes. Disinfection was quantified by counting bacteria colonies from samples aspirated before and after treatment. Histopathological evaluation of the abscesses was performed to identify changes resulting from histotripsy treatment and potential collateral damage. Cavitation histotripsy was more successful in reducing the bacterial load while having a smaller treatment volume compared with boiling histotripsy. The results of this pilot study suggest focused ultrasound may lead to a technology for in situ treatment of acoustically accessible abscesses.

Blood vessel rupture by cavitation.

Cavitation is thought to be one mechanism for vessel rupture during shock wave lithotripsy treatment. However, just how cavitation induces vessel rupture remains unknown. In this work, a high-speed photomicrography system was set up to directly observe the dynamics of bubbles inside blood vessels in ex vivo rat mesenteries. Vascular rupture correlating to observed bubble dynamics were examined by imaging bubble extravasation and dye leakage. The high-speed images show that bubble expansion can cause vessel distention, and bubble collapse can lead to vessel invagination. Liquid jets were also observed to form. Our results suggest that all three mechanisms, vessel distention, invagination and liquid jets, can contribute to vessel rupture.

Transient cavitation in high-quality-factor resonators at high static pressures.

It is well known that cavitation collapse can generate intense concentrations of mechanical energy, sufficient to erode even the hardest metals and to generate light emissions visible to the naked eye [sonoluminescence (SL)]. Considerable attention has been devoted to the phenomenon of "single bubble sonoluminescence" (SBSL) in which a single stable cavitation bubble radiates light flashes each and every acoustic cycle. Most of these studies involve acoustic resonators in which the ambient pressure is near 0.1 MPa (1 bar), and with acoustic driving pressures on the order of 0.1 MPa. This study describes a high-quality factor, spherical resonator capable of achieving acoustic cavitation at ambient pressures in excess of 30 MPa (300 bars). This system generates bursts of violent inertial cavitation events lasting only a few milliseconds (hundreds of acoustic cycles), in contrast with the repetitive cavitation events (lasting several minutes) observed in SBSL; accordingly, these events are described as "inertial transient cavitation." Cavitation observed in this high pressure resonator is characterized by flashes of light with intensities up to 1000 times brighter than SBSL flashes, as well as spherical shock waves with amplitudes exceeding 30 MPa at the resonator wall. Both SL and shock amplitudes increase with static pressure.

Effect of Stiffness of Large Extravascular Hematomas on Their Susceptibility to Boiling Histotripsy Liquefaction in Vitro.

Large intra-abdominal, retroperitoneal and intramuscular hematomas are common consequences of sharp and blunt trauma and post-surgical bleeds, and often threaten organ failure, compartment syndrome or spontaneous infection. Current therapy options include surgical evacuation and placement of indwelling drains that are not effective because of the viscosity of the organized hematoma. We have previously reported the feasibility of using boiling histotripsy (BH)-a pulsed high-intensity focused ultrasound method-for liquefaction of large volumes of freshly coagulated blood and subsequent fine-needle aspiration. The goal of this work was to evaluate the changes in stiffness of large coagulated blood volumes with aging and retraction in vitro, and to correlate these changes with the size of the BH void and, therefore, the susceptibility of the material to BH liquefaction. Large-volume (55-200 mL) whole-blood clots were fabricated in plastic molds from human and bovine blood, either by natural clotting or by recalcification of anticoagulated blood, with or without addition of thrombin. Retraction of the clots was achieved by incubation for 3 h, 3 d or 8 d. The shear modulus of the samples was measured with a custom-built indentometer and shear wave elasticity (SWE) imaging. Sizes of single liquefied lesions produced with a 1.5-MHz high-intensity focused ultrasound transducer within a 30-s standard BH exposure served as the metric for susceptibility of clot material to this treatment. Neither the shear moduli of naturally clotted human samples (0.52 ± 0.08 kPa), nor their degree of retraction (ratio of expelled fluid to original volume 50%-58%) depended on the length of incubation within 0-8 d, and were significantly lower than those of bovine samples (2.85 ± 0.17 kPa, retraction 5%-38%). In clots made from anticoagulated bovine blood, the variation of calcium chloride concentration within 5-40 mmol/L did not change the stiffness, whereas lower concentrations and the addition of thrombin resulted in significantly softer clots, similar to naturally clotted human samples. Within the achievable shear modulus range (0.4-1.6 kPa), the width of the BH-liquefied lesion was more affected by the changes in stiffness than the length of the lesion. In all cases, however, the lesions were larger compared with any soft tissue liquefied with the same BH parameters, indicating higher susceptibility of hematomas to BH damage. These results suggest that clotted bovine blood with added thrombin is an acceptable in vitro model of both acute and chronic human hematomas for assessing the efficiency of BH liquefaction strategies.

Novel ultrasmall multifunctional nanodots for dual-modal MR/NIR-II imaging-guided photothermal therapy.

Encouraging progress in multifunctional nanotheranostic agents that combine photothermal therapy (PTT) and different imaging modalities has been made. However, rational designed and biocompatible multifunctional agents that suitfable for in vivo application is highly desired but still challenging. In this work, we rationally designed novel ultrasmall multifunctional nanodots (FS-GdNDs) by combining the bovine serum albumin (BSA)-based gadolinium oxide nanodots (GdNDs) obtained through a biomineralization process with a small-molecule NIR-II fluorophore (FS). The as-prepared FS-GdNDs with an ultrasmall hydrodynamic diameter of 9.3 nm exhibited prominent NIR-II fluorescence properties, high longitudinal relaxivity (10.11 mM-1 s-1), and outstanding photothermal conversion efficiency (43.99%) and photothermal stability. In vivo studies showed that the FS-GdNDs with enhanced multifunctional characteristics diaplayed satisfactory dual-modal MR/NIR-II imaging performance with a quite low dose. The imaging-guided PTT achieved successful ablation of tumors and effectively extended the survival of mice. Cytotoxicity studies and histological assay demonstrated excellent biocompatibility of the nanodots. Importantly, this novel FS-GdNDs can undergo efficient body clearance through both hepatobiliary and renal excretion pathways. The novel ultrasmall multifunctional FS-GdNDs with excellent features hold tremendous potential in biomedical and clinical applications.

Estimating the shell parameters of SonoVue microbubbles using light scattering.

Experiments were performed to measure the dynamical response of individual SonoVue microbubbles subjected to pulsed ultrasound. Three commonly used bubble dynamic models (i.e., Hoff's, Sarkar's, and linearized Marmottant's models) were compared to determine the most appropriate model for fitting to the experimental data. The models were evaluated against published optical microscopy data. The comparison suggests that it is difficult to rank these models for lipid-shelled microbubbles undergoing small-amplitude oscillations, because under these conditions the shell parameters in these models are closely related. A linearized version of the Marmottant model was used to estimate the shell parameters (i.e., shear modulus and shear viscosity) of SonoVue microbubbles from the experimental light scattering data, as a function of ambient microbubble radius. The SonoVue microbubble shell elasticity and dilatational viscosity increase with ambient bubble radius, in agreement with previously published data. The results suggest that light scattering, used in conjunction with one of several popular bubble dynamics models, is effective at characterizing microbubble response and evaluating shell parameters.

Recent developments on foaming mechanical and electronic techniques for the management of varicose veins.

Introduction: Varicose veins are a common disease, causing significant impairment of quality of life to afflicted individuals. Conventional surgery has represented the traditional treatment for years, with significant post-operative complications. By the end of the 20th century, novel approaches had been developed to induce biochemical sclerosis into the treated vein in order to exclude it from blood circulation.Areas covered: Foaming techniques for treatment of varicose veins, both clinically-approved methods and those under experimental studies. A brief description of cavitation, which is the basis of microbubbles formation, and an overview of foam properties have been also provided, including a discussion on clinical efficacy and safety profile.Expert commentary: Foam sclerotherapy has rapidly gained popularity since it represents the most minimally invasive and cost-effective procedure in the short term. Several different methods of foam preparation have been described in literature. In general, the foam generation method may affect characteristics such as stability and bubble size distribution, which in turn affect the therapeutic action of foam itself. Therefore, the selection of a suitable foaming technique is of importance for treatment success. Future developments on foaming techniques are expected to make sclerotherapy, already an effective treatment, even safer and more versatile therapeutic procedure.

Inactivation of Planktonic Escherichia coli by Focused 1-MHz Ultrasound Pulses with Shocks: Efficacy and Kinetics Upon Volume Scale-Up.

This study addresses inactivation of E. coli in either 5- or 10-mL volumes, which were 50- to 100-fold greater than used in an earlier study (Brayman et al. 2017). Cells were treated with 1-MHz pulsed high-intensity focused ultrasound (10 cycles, 2-kHz repetition frequency, +65/-12.8 MPa focal pressures). The surviving fraction was assessed by coliform assay, and inactivation demonstrated curvilinear kinetics. The reduction of surviving fraction to 50% required 2.5 or 6 min in 5- or 10-mL samples, respectively. Exposure of 5 mL for 20 min reduced the surviving fraction to ∼1%; a similar exposure of 10-mL samples reduced the surviving fraction to ∼10%. Surviving cells from 5-min exposures appeared normal under light microscopy, with minimal debris; after 20 min, debris dominated. Transmission electron microscopy images of insonated samples showed some undamaged cells, a few damaged but largely intact cells and comminuted debris. Cellular damage associated with substantive but incomplete levels of inactivation can be variable, ranging from membrane holes tens of nanometers in diameter to nearly complete comminution.

Evaluation of a shock wave induced cavitation activity both in vitro and in vivo.

This study evaluated the cavitation activity induced by shock wave (SW) pulses, both in vitro and in vivo, based on the area measurements of echogenic regions observed in B-mode ultrasound images. Residual cavitation bubble clouds induced by SW pulses were detected as echogenic regions in B-mode images. The temporal evolution of residual bubble clouds, generated by SWs with varying lithotripter charging voltage and pulse repetition frequency (PRF), was analyzed by measuring the time-varying behaviors of the echogenic region areas recorded in B-mode images. The results showed that (1) the area of SW-induced echogenic regions enlarged with increased SW pulse number; (2) echogenic regions in the B-mode images dissipated gradually after ceasing the SWs, which indicated the dissolution of the cavitation bubbles; and (3) larger echogenic regions were generated with higher charging voltage or PRF.

Inactivation of Planktonic Escherichia coli by Focused 2-MHz Ultrasound.

This study was motivated by the desire to develop a non-invasive means to treat abscesses, and represents the first steps toward that goal. Non-thermal, high-intensity focused ultrasound (HIFU) was used to inactivate Escherichia coli (∼1 × 109 cells/mL) in suspension. Cells were treated in 96-well culture plate wells using 1.95-MHz ultrasound and incident focal acoustic pressures as high as 16 MPa peak positive and 9.9 MPa peak negative (free field measurements). The surviving fraction was assessed by coliform culture and the alamarBlue assay. No biologically significant heating was associated with ultrasound exposure. Bacterial inactivation kinetics were well described by a half-life model, with a half-time of 1.2 min. At the highest exposure levels, a 2log inactivation was typically achieved within 10 min. The free field-equivalent peak negative acoustic pressure threshold for inactivation was ∼7 MPa. At the highest acoustic pressures used, inactivation efficacy was insensitive to reciprocal changes in pulse length and pulse repetition frequency at constant duty factor. Although treated volumes were very small, proof of principle was provided by these experiments.

Inertial cavitation dose produced in ex vivo rabbit ear arteries with Optison by 1-MHz pulsed ultrasound.

Previous in vitro studies have shown that ultrasound-induced mechanical bioeffects with contrast agents present are highly correlated with inertial cavitation (IC) "dose" (Chen et al. 2003a, 2003c). The ex vivo experiments conducted here addressed the following hypotheses: 1. IC activity can be generated by insonating perfused rabbit ear blood vessel, and 2. the IC "dose" developed during insonation treatment can be reliably measured and will vary with varying acoustic parameters and Optison concentration. Ex vivo rabbit auricular arteries were perfused with Optison suspensions and then exposed to 1.1-MHz pulsed focused ultrasound. Experimental variables included peak negative acoustic pressure (0.2 MPa to 5.2 MPa), pulse-repetition frequency (5, 50 or 500 Hz), pulse length (50, 100, 500 or 1000 cycles), and Optison volume concentration (0, 0.2, 0.5 or 1%). Cavitation activity was quantified as IC dose, based on passive cavitation detection measurements. The results show that: 1. The IC pressure threshold decreases with higher concentrations of Optison, and 2. IC dose increases significantly with increasing acoustic pressure, Optison concentration, pulse length or with decreasing pulse-repetition frequency.

Intravascular inertial cavitation activity detection and quantification in vivo with Optison.

Inertial cavitation (IC) is an important mechanism by which ultrasound (US)-induced bioeffects can be produced. It has been reported that US-induced in vitro mechanical bioeffects with the presence of ultrasound contrast agents (UCAs) are highly correlated with quantified IC "dose" (ICD: cumulated root-mean-squared broadband noise amplitude in the frequency domain). The ICD has also been used to quantify IC activity in ex vivo perfused rabbit ear vessels. The in vivo experiments reported here using a rabbit ear vessel model were designed to: (1) detect and quantify IC activity in vivo within the constrained environment of rabbit auricular veins with the presence of Optison and (2) measure the temporal evolution of microbubble IC activity and the ICD generated during insonation treatment, as a function of acoustic parameters. Preselected regions-of-interest (ROI) in the rabbit ear vein were exposed to pulsed focused US (1.17 MHz, 1 Hz PRF). Experimental acoustic variables included peak rarefaction pressure amplitude ([PRPA]: 1.1, 3.0, 6.5 or 9.0 MPa) and pulse length (20, 100, 500 or 1000 cycles). ICD was quantified based on passive cavitation detection (PCD) measurements. The results show that: (1) after Optison injection, the time to onset of measurable microbubble IC activity was relatively consistent, approximately 20 s; (2) after reaching its peak value, the IC activity decayed exponentially and the half-life decay coefficient (t(1/2)) increased with increasing PRPA and pulse length; and (3) the normalized ICD generated by pulsed US exposure increased significantly with increasing PRPA and pulse length.