Efficacy of RNA interference knockdown using aerosolized short interfering RNAs bound to nanoparticles in three diverse aphid species.

RNA interference (RNAi) has emerged as a promising method for validating gene function; however, its utility in nonmodel insects has proven problematic, with delivery methods being one of the main obstacles. This study investigates a novel method of RNAi delivery in aphids, the aerosolization of short interfering RNA (siRNA)-nanoparticle complexes. By using nanoparticles as a siRNA carrier, the likelihood of cellular uptake is increased, when compared to methods previously used in insects. To determine the efficacy of this RNAi delivery system, siRNAs were aerosolized with and without nanoparticles in three aphid species: Acyrthosiphon pisum, Aphis glycines and Schizaphis graminum. The genes targeted for knockdown were carotene dehydrogenase (tor), which is important for pigmentation in Ac. pisum, and branched chain-amino acid transaminase (bcat), which is essential in the metabolism of branched-chain amino acids in all three aphid species. Overall, we observed modest gene knockdown of tor in Ac. pisum and moderate gene knockdown of bcat in Ap. glycines along with its associated phenotype. We also determined that the nanoparticle emulsion significantly increased the efficacy of gene knockdown. Overall, these results suggest that the aerosolized siRNA-nanoparticle delivery method is a promising new high-throughput and non-invasive RNAi delivery method in some aphid species.

Additional results for "joint entropy of continuously differentiable ultrasonic waveforms" [J. Acoust. Soc. Am. 133(1), 283-300 (2013)].

Previous results on the use of joint entropy for detection of targeted nanoparticles accumulating in the neovasculature of MDA435 tumors [Fig. 7 of M. S. Hughes et al., J. Acoust. Soc. Am. 133, 283-300 (2013)] are extended, with sensitivity improving by nearly another factor of 2. This result is obtained using a "quasi-optimal" reference waveform in the computation of the joint entropy imaging technique used to image the accumulating nanoparticles.

Joint entropy of continuously differentiable ultrasonic waveforms.

This study is based on an extension of the concept of joint entropy of two random variables to continuous functions, such as backscattered ultrasound. For two continuous random variables, X and Y, the joint probability density p(x,y) is ordinarily a continuous function of x and y that takes on values in a two dimensional region of the real plane. However, in the case where X=f(t) and Y=g(t) are both continuously differentiable functions, X and Y are concentrated exclusively on a curve, γ(t)=(f(t),g(t)), in the x,y plane. This concentration can only be represented using a mathematically "singular" object such as a (Schwartz) distribution. Its use for imaging requires a coarse-graining operation, which is described in this study. Subsequently, removal of the coarse-graining parameter is accomplished using the ergodic theorem. The resulting expression for joint entropy is applied to several data sets, showing the utility of the concept for both materials characterization and detection of targeted liquid nanoparticle ultrasonic contrast agents. In all cases, the sensitivity of these techniques matches or exceeds, sometimes by a factor of two, that demonstrated in previous studies that employed signal energy or alternate entropic quantities.

Theragnostics for tumor and plaque angiogenesis with perfluorocarbon nanoemulsions.

Molecular imaging agents are extending the potential of noninvasive medical diagnosis from basic gross anatomical descriptions to complicated phenotypic characterizations based upon the recognition of unique cell-surface biochemical signatures. Although originally the purview of nuclear medicine, "molecular imaging" is now studied in conjunction with all clinically relevant imaging modalities. Of the myriad of particles that have emerged as prospective candidates for clinical translation, perfluorocarbon nanoparticles offer great potential for combining targeted imaging with drug delivery, much like the "magic bullet" envisioned by Paul Ehrlich 100 years ago. Perfluorocarbon nanoparticles, once studied in Phase III clinical trials as blood substitutes, have found new life for molecular imaging and drug delivery. The particles have been adapted for use with all clinically relevant modalities and for targeted drug delivery. In particular, their intravascular constraint due to particle size provides a distinct advantage for angiogenesis imaging and antiangiogenesis therapy. As perfluorocarbon nanoparticles have recently entered Phase I clinical study, this review provides a timely focus on the development of this platform technology and its application for angiogenesis-related pathologies.

Application of Renyi entropy for ultrasonic molecular imaging.

Previous work has demonstrated that a signal receiver based on a limiting form of the Shannon entropy is, in certain settings, more sensitive to subtle changes in scattering architecture than conventional energy-based signal receivers [M. S. Hughes et al., J. Acoust. Soc. Am. 121, 3542-3557 (2007)]. In this paper new results are presented demonstrating further improvements in sensitivity using a signal receiver based on the Renyi entropy.

Real-time calculation of a limiting form of the Renyi entropy applied to detection of subtle changes in scattering architecture.

Previously a new method for ultrasound signal characterization using entropy H(f) was reported, and it was demonstrated that in certain settings, further improvements in signal characterization could be obtained by generalizing to Renyi entropy-based signal characterization I(f)(r) with values of r near 2 (specifically r=1.99) [M. S. Hughes et al., J. Acoust. Soc. Am. 125, 3141-3145 (2009)]. It was speculated that further improvements in sensitivity might be realized at the limit r-->2. At that time, such investigation was not feasible due to excessive computational time required to calculate I(f)(r) near this limit. In this paper, an asymptotic expression for the limiting behavior of I(f)(r) as r-->2 is derived and used to present results analogous to those obtained with I(f)(1.99). Moreover, the limiting form I(f,infinity) is computable directly from the experimentally measured waveform f(t) by an algorithm that is suitable for real-time calculation and implementation.

Three-dimensional characterization of human ventricular myofiber architecture by ultrasonic backscatter.

Normal human left ventricular architecture comprises a highly aligned array of cardiac myofibers whose orientation depends on transmural location. This study was designed to determine whether measurement of integrated backscatter could be used detect the progressive transmural shift of myofiber alignment that occurs from epicardium to endocardium in human ventricular wall segments. Integrated backscatter was measured at 32 transmural levels in seven cylindrical biopsy specimens (1.4 cm diam) sampled from normal regions of six explanted fixed human hearts by insonification of samples at 180 independent angles in 2 degrees steps around their entire circumference with a 5-MHz broadband piezoelectric transducer. Histologic analysis was performed to determine fiber orientation. Integrated backscatter varied approximately as a sinusoidal function of the angle of insonification at each transmural level. Greater integrated backscatter was observed for insonification perpendicular as compared with parallel to fibers (difference = 14.5 +/- 0.6 dB). Ultrasonic analysis revealed a progressive transmural shift in fiber orientation of approximately 9.2 +/- 0.7 degrees/mm of tissue. Histologic analysis revealed a concordant shift in fiber orientation of 7.9 +/- 0.8 degrees/mm of tissue. Thus, human myocardium manifests anisotropy of ultrasonic scattering that may be useful for characterization of the intramural fiber alignment and overall three-dimensional organization of cardiac myofibers.

A relationship between ultrasonic integrated backscatter and myocardial contractile function.

We have shown previously that the physiologic, mechanical cardiac cycle is associated with a parallel, cardiac cycle-dependent variation of integrated backscatter (IB). However, the mechanisms responsible are not known. The mathematical and physiological considerations explored in the present study suggest that the relationship between backscatter and myocardial contractile function reflects cyclic alterations in myofibrillar elastic parameters, with the juxtaposition of intracellular and extracellular elastic elements that have different intrinsic acoustic impedances providing an appropriately sized scattering interface at the cellular level. Cardiac cycle-dependent changes in the degree of local acoustic impedance mismatch therefore may elicit concomitant changes in backscatter. Because acoustic impedance is determined partly by elastic modulus, changes in local elastic moduli resulting from the non-Hookian behavior of myocardial elastic elements exposed to stretch may alter the extent of impedance mismatch. When cardiac cell mechanical behavior is represented by a three-component Maxwell-type model of muscle mechanics, the systolic decrease in IB that we have observed experimentally is predicted. Our prior observations of regional intramural differences in IB and the dependence of IB on global contractile function are accounted for as well. When the model is tested experimentally by assessing its ability to predict the regional and global behavior of backscatter in response to passive left ventricular distention, good concordance is observed.

Properties of an entropy-based signal receiver with an application to ultrasonic molecular imaging.

Qualitative and quantitative properties of the finite part, H(f), of the Shannon entropy of a continuous waveform f(t) in the continuum limit are derived in order to illuminate its use for waveform characterization. Simple upper and lower bounds on H(f), based on features of f(t), are defined. Quantitative criteria for a priori estimation of the average-case variation of H(f) and log E(f), where E(f) is the signal energy of f(t) are also derived. These provide relative sensitivity estimates that could be used to prospectively choose optimal imaging strategies in real-time ultrasonic imaging machines, where system bandwidth is often pushed to its limits. To demonstrate the utility of these sensitivity relations for this application, a study designed to assess the feasibility of identification of angiogenic neovasculature targeted with perfluorocarbon nanoparticles that specifically bind to alpha(v)beta3-integrin expression in tumors was performed. The outcome of this study agrees with the prospective sensitivity estimates that were used for the two receivers. Moreover, these data demonstrate the ability of entropy-based signal receivers when used in conjunction with targeted nanoparticles to elucidate the presence of alpha(v)beta3 integrins in primordial neovasculature, particularly in acoustically unfavorable environments.

Occult cardiac contractile dysfunction in dystrophin-deficient children revealed by cardiac magnetic resonance strain imaging.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an inherited disease characterized by early onset of skeletal muscle degeneration and progressive weakness. Although dilated cardiomyopathy may occur during adolescence, it is often undetected early in its course because of physical inactivity and generalized debilitation. The purpose of this study was to apply the technique of cardiac magnetic resonance (CMR) tagging to detect occult cardiac dysfunction in young subjects with DMD by measuring myocardial strain and torsion. METHODS AND RESULTS: Thirteen DMD pediatric subjects without clinically apparent heart disease and 9 age-matched healthy males were recruited. Each was scanned on a 1.5-T clinical scanner to acquire contiguous short-axis planes from the apex to the mitral valve plane and then 3 tagged images at base, midventricle, and apex. Global and segmental myocardial net twist and circumferential strain were computed with the use of 2D homogeneous strain analysis. Ventricular torsion was computed by normalizing net twist by the distance from apex to mitral valve plane. DMD patients exhibited normal left ventricular volumes and ejection fractions but manifested reduced midventricular and basal cross-sectional global circumferential strain compared with the reference group (P<0.005). These alterations also appeared in segmental analyses in the septal, anterior, lateral, and inferior walls (P<0.05). CONCLUSIONS: In patients predisposed to cardiomyopathies because of dystrophinopathy, occult regional cardiac dysfunction can be diagnosed with CMR tagging. This method of strain imaging analysis may offer a sensitive approach for delineating the presence and progression of cardiovascular disease and for assessing therapies designed to modulate the onset and course of heart failure.

Novel MRI contrast agent for molecular imaging of fibrin: implications for detecting vulnerable plaques.

BACKGROUND: Molecular imaging of thrombus within fissures of vulnerable atherosclerotic plaques requires sensitive detection of a robust thrombus-specific contrast agent. In this study, we report the development and characterization of a novel ligand-targeted paramagnetic molecular imaging agent with high avidity for fibrin and the potential to sensitively detect active vulnerable plaques. METHODS AND RESULTS: The nanoparticles were formulated with 2.5 to 50 mol% Gd-DTPA-BOA, which corresponds to >50 000 Gd(3+) atoms/particle. Paramagnetic nanoparticles were characterized in vitro and evaluated in vivo. In contradistinction to traditional blood-pool agents, T1 relaxation rate as a function of paramagnetic nanoparticle number was increased monotonically with Gd-DTPA concentration from 0.18 mL. s(-1). pmol(-1) (10% Gd-DTPA nanoparticles) to 0.54 mL. s(-1). pmol(-1) for the 40 mol% Gd-DTPA formulations. Fibrin clots targeted in vitro with paramagnetic nanoparticles presented a highly detectable, homogeneous T1-weighted contrast enhancement that improved with increasing gadolinium level (0, 2.5, and 20 mol% Gd). Higher-resolution scans and scanning electron microscopy revealed that the nanoparticles were present as a thin layer over the clot surface. In vivo contrast enhancement under open-circulation conditions was assessed in dogs. The contrast-to-noise ratio between the targeted clot (20 mol% Gd-DTPA nanoparticles) and blood was approximately 118+/-21, and that between the targeted clot and the control clot was 131+/-37. CONCLUSIONS: These results suggest that molecular imaging of fibrin-targeted paramagnetic nanoparticles can provide sensitive detection and localization of fibrin and may allow early, direct identification of vulnerable plaques, leading to early therapeutic decisions.

Inhibition of thrombin attenuates stenosis after arterial injury in minipigs.

OBJECTIVES: We sought to determine whether brief, profound inhibition of thrombin or prothrombin activation by factor Xa limits neointimal formation and stenosis after arterial injury. BACKGROUND: Thrombin has been implicated as a mediator of neointimal formation, but adjunctive administration of anticoagulant agents has not proven effective to decrease restenosis in patients undergoing coronary angioplasty. METHODS: We infused recombinant desulfatohirudin (r-hirudin, bolus of 2 mg/kg body weight followed by 2 mg/kg per h, n = 9), heparin (100 U/kg per h, n = 6) or recombinant tick anticoagulant peptide (rTAP, 1-mg/kg bolus followed by 3 mg/kg per h, n = 5), a specific inhibitor of factor Xa, intravenously, beginning 15 min before and for up to 3 h after repetitive balloon hyperinflations sufficient to disrupt the internal elastic lamina in a carotid artery of minipigs with hypercholesterolemia induced by feeding them an atherogenic diet. RESULTS: Partial thromboplastin time was increased six- to sevenfold over baseline levels at the end of the infusions of the anticoagulant agents. Lumen stenosis measured histologically 4 weeks after balloon-induced carotid injury was 29 +/- 16% (mean +/- SEM) in r-hirudin-treated, 52 +/- 19% in rTAP-treated and 76 +/- 18% in heparin-treated pigs (p < 0.02 for r-hirudin vs. heparin treatment). CONCLUSIONS: The marked reduction of stenosis in r-hirudin-treated animals indicates that thrombin plays a major role in neointimal formation after balloon-induced arterial injury. A relatively brief interval of profound, direct inhibition of thrombin may be particularly effective to attenuate restenosis after balloon angioplasty.

Early identification with ultrasonic integrated backscatter of viable but stunned myocardium in dogs.

It has been shown that canine and human hearts exhibit a cardiac cycle-dependent variation of integrated backscatter (cyclic variation) that reflects intrinsic regional contractile performance. To determine whether ultrasound tissue characterization can identify viable though stunned myocardium before recovery of regional wall thickening, transient ischemic injury was produced in eight open chest dogs for 15 min followed by reperfusion for 2 h. Cyclic variation and wall thickening were measured before ischemia, at 15 min after the onset of ischemia and at selected intervals after the onset of reperfusion from multiple sites within the ischemic zone with a novel combined two-dimensional and M-mode acquisition system. Cyclic variation and wall thickening were computed from digitized M-mode integrated backscatter images with an algorithm developed and validated for this purpose. Magnitude and "delay" of cyclic variation and wall thickening were compared. Delay represents the degree of synchrony of regional cyclic variation or wall thickening with global ventricular mechanical systole. Baseline cyclic variation and wall thickening magnitudes were 3.8 +/- 0.2 dB and 37 +/- 1.4%, respectively. With ischemia, cyclic variation and wall thickening decreased to 1.7 +/- 0.2 dB and 17 +/- 2%, respectively (p less than 0.05, compared with baseline). Cyclic variation recovered to baseline levels within 20 min after reperfusion (3.3 +/- 0.4 dB, p = NS). Wall thickening remained depressed for 2 h after the onset of reperfusion (23 +/- 2%, p less than 0.05 compared with baseline). Delay of cyclic variation in a unitless ratio expressed as delay (in milliseconds) divided by the QT interval (in milliseconds) increased from 0.87 +/- 0.03 at baseline to 1.10 +/- 0.12 with ischemia, a change consistent with mild asynchrony, and returned to baseline (0.95 +/- 0.07, p = NS compared with baseline) within 20 min after reperfusion. Delay of wall thickening was 0.88 +/- 0.02 at baseline, increased to 1.23 +/- 0.09 with ischemia and remained significantly increased 2 h after reperfusion (1.07 +/- 0.05, p less than 0.05 compared with baseline). Recovery time constants for cyclic variation and wall thickening with reperfusion reflected earlier restoration of cyclic variation (8.1 min) than of wall thickening (420.5 min). Thus, cyclic variation recovers before wall thickening with reperfusion. Its analysis appears to provide a useful index of the presence of viable and potentially salvageable tissue in regions of stunned myocardium that is independent of wall thickening.

Ultrasound integrated backscatter tissue characterization of remote myocardial infarction in human subjects.

To determine whether quantitative ultrasound tissue characterization differentiates normal myocardial regions from segments of remote infarction, 32 consecutive patients with a diagnosis of previous myocardial infarction were evaluated. Images were obtained in real time with a modified two-dimensional ultrasound system capable of providing continuous signals in proportion to the logarithm of integrated backscatter along each A line. In 15 patients, adequate parasternal long-axis images that delineated both normal and infarct segments were obtained with standard time-gain compensation. Image data were analyzed to yield both magnitude and delay (electrocardiographic R wave to nadir normalized for the QT interval) of the cyclic variation of backscatter. Cyclic variation was present in 55 of 56 normal myocardial sites, averaging (mean +/- SEM) 3.2 +/- 0.2 dB in magnitude and exhibiting a mean normalized delay of 0.87 +/- 0.03. The magnitude of cyclic variation in infarct segments was significantly reduced to 1.1 +/- 0.2 dB (42 sites), and the delay was markedly increased to 1.47 +/- 0.12 (21 sites) (p less than 0.0001 for both). In 20 of 42 infarct sites, no cyclic variation was detectable. Thus, ultrasound tissue characterization quantitatively differentiated infarct segments from normal myocardium in patients with remote myocardial infarction.

Advances in ultrasound methods for high-resolution imaging of the cardiovascular system.

Acoustic microscopy entails the use of high-frequency high-resolution ultrasound methods to produce images of sound waves reflected from or propagated through some tissue of interest. The image contrast depends on microscopic differences in the intrinsic material properties of the substance imaged, such as mass density or compressibility. Pathologic changes in cardiovascular tissues at the subcellular level can be observed with high-frequency acoustic imaging techniques, based on alterations in the structure, properties, and organization of cells and their surrounding matrix. Potential applications extend from delineation of cardiovascular development in experimental animals to clinical characterization of the composition of atherosclerotic lesions with intravascular ultrasound and estimation of the potential for plaque rupture and infarction. (Trends Cardiovasc Med 1997;7:168-174). © 1997, Elsevier Science Inc.

Quantitative ultrasonic imaging: tissue characterization and instantaneous quantification of cardiac function.

Quantitative myocardial tissue characterization is being developed to complement and expand conventional echocardiography by delineating the physical state of myocardium under diverse pathophysiologic conditions. Real-time quantitative integrated backscatter imaging has already been applied to patients with ischemic heart disease, hypertrophic cardiomyopathy, and cardiac allograft rejection in clinical investigations performed in the United States, Europe, and Japan. A recently introduced modification of imaging processing algorithms employed for characterization of tissue facilitates automatic detection of endocardial-blood interfaces and on-line quantification of ventricular size and function. Further progress and anticipated developments in quantitative ultrasonic imaging will undoubtedly augment the clinical applications of tissue characterizations based on myocardial integrated backscatter for improved diagnosis, elucidation of pathophysiology, and assessment of cardiac function.

Molecular imaging of stretch-induced tissue factor expression in carotid arteries with intravascular ultrasound.

RATIONALE AND OBJECTIVES: Molecular imaging with targeted contrast agents enables tissues to be distinguished by detecting specific cell-surface receptors. In the present study, a ligand-targeted acoustic nanoparticle system is used to identify angioplasty-induced expression of tissue factor by smooth muscle cells within carotid arteries. METHODS: Pig carotid arteries were overstretched with balloon catheters, treated with tissue factor-targeted or a control nanoparticle system, and imaged with intravascular ultrasound before and after treatment. RESULTS: Tissue factor-targeted emulsions bound and increased the echogenicity and gray-scale levels of overstretched smooth muscle cells within the tunica media, versus no change in contralateral control arteries. Expression of stretch-induced tissue factor in carotid artery media was confirmed by immunohistochemistry. CONCLUSIONS: The potential for abnormal thrombogenicity of balloon-injured arteries, as reflected by smooth muscle expression of tissue factor, was imaged using a novel, targeted, nanoparticulate ultrasonic contrast agent.

Captopril therapy limits ventricular remodeling but does not alter myocardial collagen fiber morphology of cardiomyopathic hamsters.

Previous studies of the effect of angiotensin-converting enzyme (ACE) inhibitors on the process of cardiac remodeling have devoted little attention to potentially beneficial alterations in collagen fiber morphology and microscopic organization. The present work is part of a continuing effort to define mechanisms responsible for changes in microscopic material properties of cardiac tissue that are induced by such pharmacologic therapy. Morphologic evaluation of 11 cardiomyopathic (CM) and 5 control hamsters was performed. Six CM hamsters received captopril for 3 months in their drinking water (2 gm/l), and five other CM hamsters and five normal control hamsters received no treatment. Myocyte and collagen content, organization, and fiber size were defined with the use of circular statistics in fixed sections that were stained with picrosirius red and viewed with polarized light. The scar regions from both treated and untreated CM hearts manifested similar collagen fiber thicknesses, organization (angular deviation 21.1 ± 0.7 degrees vs. 19.2 ± 2.2 degrees, untreated vs. treated, p = NS), and content (65.0% ± 2.2% vs. 65.7% ± 3.7%, untreated vs. treated, p = NS). However, significant muscle fiber disarray was observed in myocytes in the non-necrotic zones near scars for both treated and untreated CM heart, and a strong trend toward normalization of myocyte alignment was observed after captopril therapy. In the present study, captopril exerted no significant effect on collagen content, two-dimensional fiber organization, or fiber thickness in either scar or nonscar regions. Thus, the beneficial effects of captopril on cardiac material properties in ventricular remodeling associated with heritable cardiomyopathy does not appear to be related to alterations in collagen fiber morphology or organization. However, the trend toward normalization of myocyte alignment induced by captopril in non-necrotic zones suggests a possible mechanism for the known beneficial effects of captopril on favorable ventricular remodeling.

Oxygenation of the cerebral and coronary circulation with right axillary artery perfusion during venoarterial bypass in primates.

The effectiveness of right axillary artery perform in delivering oxygenated blood to the cerebral and coronary circulation during venoarterial bypass in primates was studied. Both right and left common carotid flow measurements and arterial gas measurements revealed high flows and elevated PO2 levels. Incomplete mixing in the ascending aorta was observed from cineangiograms taken at various pump oxygenator flows in 1 animal. The results demonstrated that the brain receives excellent oxygenation at all bypass levels. However, the coronary circulation is perfused primarily by blood ejected from the left ventricle and receives only minimal contribution of well-oxygenated blood from the pump oxygenator circuit. Therefore, the heart may suffer prolonged hypoxemia during long-term venoarterial bypass for acute respiratory insufficiency.