This contribution demonstrates that quantitative ultrasound (QUS) capabilities are platform independent, using an in vivo model. Frequency-dependent attenuation estimates, backscatter coefficient, and effective scatterer diameter estimates are shown to be comparable across four different ultrasound imaging systems with varied processing techniques. The backscatter coefficient (BSC) is a fundamental material property from which several QUS parameters are estimated; therefore, consistent BSC estimates among different systems must be demonstrated. This study is an intercomparison of BSC estimates acquired by three research groups (UIUC, UW, ISU) from four in vivo spontaneous rat mammary fibroadenomas using three clinical array systems and a single-element laboratory scanner system. Because of their highly variable backscatter properties, fibroadenomas provided an extreme test case for BSC analysis, and the comparison is across systems for each tumor, not across the highly heterogeneous tumors. RF echo data spanning the 1 to 12 MHz frequency range were acquired in three dimensions from all animals using each system. Each research group processed their RF data independently, and the resulting attenuation, BSC, and effective scatterer diameter (ESD) estimates were compared. The attenuation estimates across all systems showed the same trends and consistently fit the power-law dependence on frequency. BSCs varied among the multiple slices of data acquired by each transducer, with variations between transducers being of a similar magnitude as those from slice to slice. Variation between BSC estimates was assessed via functional signal-to-noise ratios derived from back-scatter data. These functional signal-to-noise ratios indicated that BSC versus frequency variations between systems ranged from negligible compared with the noise level to roughly twice the noise level. The corresponding functional analysis of variance (fANOVA) indicated statistically significant differences between BSC curves from different systems. However, root mean squared difference errors of the BSC values (in decibels) between different transducers and imaging platforms were less than half of the BSC magnitudes in most cases. Statistical comparison of the effective scatterer diameter (ESD) estimates resulted in no significant differences in estimates from three of the four transducers used for those estimates, demonstrating agreement among estimates based on the BSC. This technical advance demonstrates that these in vivo measurements can be made in a system-independent manner; the necessary step toward clinical implementation of the technology.
Human lens lipid membranes prepared using a rapid solvent exchange method from the total lipids extracted from the clear lens cortex and nucleus of 41- to 60-year-old donors were investigated using electron paramagnetic resonance spin-labeling. Profiles of the phospholipid alkyl-chain order, fluidity, oxygen transport parameter, and hydrophobicity were assessed across coexisting membrane domains. Membranes prepared from the lens cortex and nucleus were found to contain two distinct lipid environments, the bulk phospholipid-cholesterol domain and the cholesterol bilayer domain (CBD). The alkyl chains of phospholipids were strongly ordered at all depths, indicating that the amplitude of the wobbling motion of alkyl chains was small. However, profiles of the membrane fluidity, which explicitly contain time (expressed as the spin-lattice relaxation rate) and depend on the rotational motion of spin labels, show relatively high fluidity of alkyl chains close to the membrane center. Profiles of the oxygen transport parameter and hydrophobicity have a rectangular shape and also indicate a high fluidity and hydrophobicity of the membrane center. The amount of CBD was greater in nuclear membranes than in cortical membranes. The presence of the CBD in lens lipid membranes, which at 37°C showed a permeability coefficient for oxygen about 60% smaller than across a water layer of the same thickness, would be expected to raise the barrier for oxygen transport across the fiber cell membrane. Properties of human membranes are compared with those obtained for membranes made of lipids extracted from cortex and nucleus of porcine and bovine eye lenses.
cholesterol; membrane domains; fluidity; hydrophobic barrier; oxygen permeation; EPR; spin labeling
For the first time, and using an acoustical method, it has been shown experimentally that the inertial cavitation threshold pressure of an albumin-shelled microbubble is significantly correlated with its initial size.
The cholesterol-fed rabbit is useful for atherosclerosis research. We describe development of a low-magnesium (Mg) cholesterol-containing diet to accelerate atherosclerosis in this model. Male New Zealand White rabbits were fed either chow or one of four atherogenic diets: 1% cholesterol 10% fat 0.11% Mg, 1% cholesterol 10% fat 0.40% Mg, 2% cholesterol 20% fat 0.11% Mg, or 2% cholesterol 20% fat 0.40% Mg. While feed intake decreased in cholesterol-fed rabbits, they were able to maintain their body weights. Rabbits consuming cholesterol experienced profound hypercholesterolemia and tissue lipid accumulation, with plasma cholesterol levels above 1500 mg/dl for all groups at the completion of the study. Liver and spleen lipid content and liver cholesterol content also increased. Aortic arch atheroma thickness was greatest in 1% cholesterol 10% fat 0.11% Mg animals. Tissue Mg levels decreased in cholesterol-fed animals compared to chow-fed controls, despite equal or greater serum Mg levels. Our results indicate that the 1% cholesterol 10% fat 0.11% Mg diet was optimal at promoting hypercholesterolemia and atherosclerosis while minimizing health complications for the animals. The low Mg cholesterol diet will be useful to other biomedical researchers interested in utilizing the rabbit for cardiovascular disease research.
atherosclerosis; cardiovascular diseases; cholesterol; magnesium; rabbits
Quantify the effects of microbubble (MB) size, elasticity and pulsed ultrasonic parameters on in vitro sonothrombolysis (STBL: ultrasound-mediated thrombolysis) efficacy.
MATERIALS AND METHODS
Monodispersive MBs with diameters of 1 μm or 3 μm were exposed to pulsed ultrasound (1 MHz or 3 MHz) to lyse rabbit blood clots. STBL efficacy (clot mass loss) was measured as functions of MB size and concentration, ultrasonic frequency and intensity, pulse duration (PD), pulse repeat frequency (PRF) and duty factor.
STBL at 1 MHz was more effective using 3 μm MBs and at 3 MHz using 1 μm MBs. STBL was generally more effective when 75% or more of MBs remained intact, especially for 3 μm MBs; and improving STBL by increasing PRF from 100 Hz to 400 Hz at 3 MHz was associated with increasing 3 μm MB survival. However, 60% of 1 μm MBs were destroyed during maximal STBL at 3 MHz, indicating that considerable MB collapse may be requisite for STBL under these conditions.
The ability to control MB size and elasticity permits utilizing a wide range of ultrasound parameters (frequency, intensity, etc.) to produce desired levels of STBL. Comparable, maximal STBL efficacy was achieved at twenty-fold lower intensity with 3 μm MBs (0.1 W/cm2) than with 1 μm MBs (2.0 W/cm2); a potential safety issue for in vivo STBL. Ultrasound parameters that maximized MB survival yielded maximal STBL efficacy; except with 1 μm MBs at 3 MHz where most MBs were destroyed.
sonothrombolysis; microbubble; ultrasound; cavitation
The objective of this study is to investigate cardiac bioeffects resulting from ultrasonic stimulation using a specific set of acoustical parameters. Ten Sprague–Dawley rats were anesthetized and exposed to 1-MHz ultrasound pulses of 3-MPa peak rarefactional pressure and approximately 1% duty factor. The pulse repetition frequency started slightly above the heart rate and was decreased by 1 Hz every 10 s, for a total exposure duration of 30 s. The control group was composed of five rats. Two-way analysis of variance for repeated measures and Bonferroni post hoc tests were used to compare heart rate and ejection fraction, which was used as an index of myocardial contractility. It was demonstrated for the first time that transthoracic ultrasound has the potential to decrease the heart rate by ~20%. The negative chronotropic effect lasted for at least 15 min after ultrasound exposure and there was no apparent gross damage to the cardiac tissue.
Cellular proteins are essential for human immunodeficiency virus type 1 (HIV-1) replication and may serve as viable new targets for treating infection. Using gene trap insertional mutagenesis, a high-throughput approach based on random inactivation of cellular genes, candidate genes were found that limit virus replication when mutated. Disrupted genes (N=87) conferring resistance to lytic infection with several viruses were queried for an affect on HIV-1 replication by utilizing small interfering RNA (siRNA) screens in TZM-bl cells. Several genes regulating diverse pathways were found to be required for HIV-1 replication, including DHX8, DNAJA1, GTF2E1, GTF2E2, HAP1, KALRN, UBA3, UBE2E3, and VMP1. Candidate genes were independently tested in primary human macrophages, toxicity assays, and/or Tat-dependent β-galactosidase reporter assays. Bioinformatics analyses indicated that several host factors present in this study participate in canonical pathways and functional processes implicated in prior genome-wide studies. However, the genes presented in this study did not share identity with those found previously. Novel antiviral targets identified in this study should open new avenues for mechanistic investigation.
Estimating total ultrasound attenuation from backscatter data is essential in the field of quantitative ultrasound (QUS) because of the need to compensate for attenuation when estimating the backscatter coefficient and QUS parameters. This work uses a reference phantom method of attenuation estimation to create a spatial map of attenuation slope (AS) from backscatter radio-frequency (RF) data of three phantoms and a rat mammary adenocarcinoma tumor (MAT). The attenuation maps show changes in attenuation between different regions of the phantoms and the MAT tumor. Analyses of the attenuation maps of the phantoms suggest that the AS estimates are in good quantitative agreement with the known values for the phantoms. Furthermore, estimates of total attenuation from the attenuation maps are likewise in good quantitative agreement with known values.
attenuation; attenuation slope; total attenuation; parametric image; quantitative ultrasound imaging; ultrasound
A hypometabolic state can be induced in mice by 5′-AMP administration. Previously we proposed that an underlying mechanism for this hypometabolism is linked to reduced erythrocyte oxygen transport function due to 5′-AMP uptake altering the cellular adenylate equilibrium. To test this hypothesis, we generated mice deficient in adenosine monophosphate deaminase 3 (AMPD3), the key catabolic enzyme for 5′-AMP in erythrocytes. Mice deficient in AMPD3 maintained AMPD activities in all tissues except erythrocytes. Developmentally and morphologically, the Ampd3−/− mice were indistinguishable from their wild type siblings. The levels of ATP, ADP but not 5′-AMP in erythrocytes of Ampd3−/− mice were significantly elevated. Fasting blood glucose levels of the Ampd3−/− mice were comparable to wild type siblings. In comparison to wild type mice, the Ampd3−/− mice displayed a deeper hypometabolism with a significantly delayed average arousal time in response to 5′-AMP administration. Together, these findings demonstrate a central role of AMPD3 in the regulation of 5′-AMP mediated hypometabolism and further implicate erythrocytes in this behavioral response.
Backscatter and attenuation coefficient estimates are needed in many quantitative ultrasound strategies. In clinical applications, these parameters may not be easily obtained because of variations in scattering by tissues overlying a region of interest (ROI). The goal of this study is to assess the accuracy of backscatter and attenuation estimates for regions distal to nonuniform layers of tissue-mimicking materials. In addition, this work compares results of these estimates for “layered” phantoms scanned using different clinical ultrasound machines. Two tissue-mimicking phantoms were constructed, each exhibiting depth-dependent variations in attenuation or backscatter. The phantoms were scanned with three ultrasound imaging systems, acquiring radio frequency echo data for offline analysis. The attenuation coefficient and the backscatter coefficient (BSC) for sections of the phantoms were estimated using the reference phantom method. Properties of each layer were also measured with laboratory techniques on test samples manufactured during the construction of the phantom. Estimates of the attenuation coefficient versus frequency slope, α0, using backscatter data from the different systems agreed to within 0.24 dB/cm-MHz. Bias in the α0 estimates varied with the location of the ROI. BSC estimates for phantom sections whose locations ranged from 0 to 7 cm from the transducer agreed among the different systems and with theoretical predictions, with a mean bias error of 1.01 dB over the used bandwidths. This study demonstrates that attenuation and BSCs can be accurately estimated in layered inhomogeneous media using pulse-echo data from clinical imaging systems.
attenuation; backscatter; ultrasound; inhomogeneity; quantitative ultrasound; tissue-mimicking phantom; interlaboratory; comparison
Quantitative ultrasound (QUS) techniques that parameterize the backscattered power spectrum have demonstrated significant promise for ultrasonic tissue characterization. Some QUS parameters, such as the effective scatterer diameter (ESD), require the assumption that the examined medium contains uniform diffuse scatterers. Structures that invalidate this assumption can significantly affect the estimated QUS parameters and decrease performance when classifying disease. In this work, a method was developed to reduce the effects of echoes that invalidate the assumption of diffuse scattering. To accomplish this task, backscattered signal sections containing non-diffuse echoes were identified and removed from the QUS analysis. Parameters estimated from the generalized spectrum (GS) and the Rayleigh SNR parameter were compared for detecting data blocks with non-diffuse echoes. Simulations and experiments were used to evaluate the effectiveness of the method. Experiments consisted of estimating QUS parameters from spontaneous fibroadenomas in rats and from beef liver samples. Results indicated that the method was able to significantly reduce or eliminate the effects of non-diffuse echoes that might exist in the backscattered signal. For example, the average reduction in the relative standard deviation of ESD estimates from simulation, rat fibroadenomas, and beef liver samples were 13%, 30%, and 51%, respectively. The Rayleigh SNR parameter performed best at detecting non-diffuse echoes for the purpose of removing and reducing ESD bias and variance. The method provides a means to improve the diagnostic capabilities of QUS techniques by allowing separate analysis of diffuse and non-diffuse scatterers.
The organization and physical properties of the lipid bilayer portion of intact cortical and nuclear fiber cell plasma membranes isolated from the eyes lenses of two-year-old pigs were studied using electron paramagnetic resonance (EPR) spin-labeling. Membrane fluidity, hydrophobicity, and the oxygen transport parameter (OTP) were assessed from the EPR spectra of precisely positioned spin labels. Intact cortical and nuclear membranes, which include membrane proteins, were found to contain three distinct lipid environments. These lipid environments were termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain (lipids in protein aggregates). The amount of boundary and trapped lipids was greater in intact nuclear membranes than in cortical membranes. The properties of intact membranes were compared with the organization and properties of lens lipid membranes made of the total lipid extracts from the lens cortex or nucleus. In cortical lens lipid membranes, only one homogenous environment was detected, which was designated as a bulk lipid domain (phospholipid bilayer saturated with cholesterol). Lens lipid membranes prepared from the lens nucleus possessed two domains, assigned as a bulk lipid domain and a cholesterol bilayer domain (CBD). In intact nuclear membranes, it was difficult to discriminate the CBD, which was clearly detected in nuclear lens lipid membranes because the OTP measured in the CBD is the same as in the domain formed by trapped lipids. The two domains unique to intact membranes—namely, the domain formed by boundary lipids and the domain formed by trapped lipids—were most likely formed due to the presence of membrane proteins. It is concluded that formation of rigid and practically impermeable domains is enhanced in the lens nucleus, indicating changes in membrane composition that may help to maintain low oxygen concentration in this lens region.
cholesterol; membrane domains; fluidity; hydrophobic barrier; oxygen permeation; EPR; spin labeling
Uniformly-sized preparations with average microbubble (MB) diameters from 1 µm to 7 µm were produced reliably by sonicating decafluorobutane-saturated solutions of serum albumin and dextrose. Detailed protocols for producing and size-separating the MBs are presented, along with the effects that changing each production parameter (serum albumin concentration, sonication power, sonication time, etc.) had on MB size distribution and acoustic stability. These protocols can be used to produce MBs for experimental applications or serve as templates for developing new protocols that yield MBs with physical and acoustic properties better suited to specific applications. Size stability and ultrasonic performance quality control tests were developed to assure that successive MB preparations perform identically and to distinguish the physical and acoustic properties of identically sized MBs produced with different serum albumin-dextrose formulations and sonication parameters. MBs can be stored at 5°C for protracted periods (2 weeks to one year depending on formulation).
microbubble; ultrasound; contrast agent; production; sonication; cavitation
Nitric Oxide (NO) plays a critical role in diverse physiological and pathological processes. We show that a hypomorphic mouse model of argininosuccinate lyase (Asl) deficiency exhibits a distinct phenotype manifest by multi-organ dysfunction and NO deficiency. Loss of Asl leads to reduced NO synthesis due to decreased endogenous arginine synthesis as well as reduced utilization of extracellular arginine for NO production in both humans and mice. Hence, ASL as seen in other species through evolution has a structural function in addition to its catalytic activity. Importantly, therapy with nitrite rescued the tissue autonomous NO deficiency in hypomorphic Asl mice, while a NOS independent NO donor restored NO-dependent vascular reactivity in subjects with ASL deficiency. Our data demonstrate a previously unappreciated role for ASL in NOS function and NO homeostasis. Hence, ASL may serve as a target for manipulating NO production in experimental models, as well as treatment of NO-related diseases.
Ultrasound contrast agents (UCAs) are intravenously infused microbubbles that add definition to ultrasonic images. Ultrasound contrast agents continue to show clinical promise in cardiovascular imaging, but their biological effects are not known with confidence. We used a cholesterol-fed rabbit model to evaluate these effects when used in conjunction with ultrasound (US) to image the descending aorta.
Male New Zealand White rabbits (n = 41) were weaned onto an atherogenic diet containing 1% cholesterol, 10% fat, and 0.11% magnesium. At 21 days, rabbits were exposed to contrast US at 1 of 4 pressure levels using either the UCA Definity (Lantheus Medical Imaging, Inc, North Billerica, MA) or a saline control (n = 5 per group). Blood samples were collected and analyzed for lipids and von Willebrand factor (vWF), a marker of endothelial function. Animals were euthanized at 42 days, and tissues were collected for histologic analysis.
After adjustment for pre-exposure vWF, high-level US (in situ [at the aorta] peak rarefactional pressure of 1.4 or 2.1 MPa) resulted in significantly lower vWF 1 hour post exposure (P = .0127; Padj < .0762). This difference disappeared within 24 hours. Atheroma thickness in the descending aorta was lower in animals receiving the UCA compared to animals receiving saline.
Contrast US affected the descending aorta, as evidenced by two separate outcome measures. These results may be a first step in elucidating a previously unknown biological effect of UCAs. Further research is warranted to characterize the effects of this procedure.
analysis of covariance; atherosclerosis; biomarkers; contrast media; endothelium; microbubbles; ultrasound
Gamunex 10% (Talecris Biotherapeutics, Research Triangle Park, NC), a commercially available preparation of pooled human immunoglobulin G, has been proposed as an antitoxin therapy against bacterial toxins released in infectious endophthalmitis. Its biocompatibility with two commonly used intraocular infusion fluids was evaluated to determine feasibility of its clinical application in endophthalmitis treatment.
Gamunex 10% was mixed with BSS or BSS Plus (Alcon Laboratories, Fort Worth, TX) such that it constituted a range of 1.25%–50% by volume. Osmolality, pH, optical density, and ionic strength were measured across this range of concentrations.
The amount of pH reduction with increasing concentrations of Gamunex 10% was similar for both BSS and BSS Plus. In BSS Plus, solutions containing up to 20% by volume of Gamunex 10% remained at near-physiologic pH (∼7.0 or above). No physiologically significant changes in osmolality or optical density measurements that would be anticipated to have profound physiological effects were observed at any of the measured concentrations, nor was there visual evidence of tubidity/precipitation. A gradual increase in ionic strength was observed with increasing concentrations of Gamunex 10%.
Potentially therapeutic mixtures of Gamunex 10% in 2 commonly used intraocular infusion fluids, BSS and BSS Plus, showed no evidence of bioincompatibility when the solutions were evaluated for changes in osmolality, pH, ionic strength, aggregation, or precipitation.
The interaction of ultrasound contrast agents (UCAs) and ultrasound (US) provides a way to spatially and temporally target tissues. Recently, UCAs have been used therapeutically to induce localized angiogenesis. Ultrasound contrast agents, however, have been documented to induce negative bioeffects. To further understand the balance of risks and benefits of UCAs and to examine the mechanism of US-UCA–induced angiogenesis, this study explored the role of UCAs, in particular Definity (Lantheus Medical Imaging, Inc, North Billerica, MA), in producing an angiogenic response.
The gracilis muscles of Sprague Dawley rats were exposed to 1-MHz US. The rats were euthanized the same day or allowed to recover for 3 or 6 days post exposure (DPE). Ultrasound peak rarefactional pressures (Prs) of 0.25, 0.83, 1.4, and 2.0 MPa were used while rats were infused with either saline or Definity. Assessments for angiogenesis included capillary density, inflammation, and vascular endothelial growth factor (VEGF), both acutely (0 DPE) and at 3 and 6 DPE.
The results of this study suggest that the angiogenic response is dependent on infusion media, Pr, and DPE. While capillary density did not reach significance, VEGF expression was significant for infusion media, Pr, and DPE with inflammation co-occurrence (P < .05).
These results suggest that the angiogenic response is elicited by a mechanical effect of US-UCA stimulation of VEGF that is potentially optimized when collapse occurs.
angiogenesis; proangiogenic therapy; therapeutic ultrasound; ultrasound contrast agent; ultrasound-induced bioeffects; vascular endothelial growth factor
In vivo estimations of the frequency-dependent acoustic attenuation (α) and backscatter (η) coefficients using radio frequency (RF) echoes acquired with clinical ultrasound systems must be independent of the data acquisition setup and the estimation procedures. In a recent in vivo assessment of these parameters in rodent mammary tumors, overall agreement was observed among α and η estimates using data from four clinical imaging systems. In some cases, particularly in highly attenuating heterogeneous tumors, multi-system variability was observed. This paper compares α and η estimates of a well-characterized rodent-tumor-mimicking homogeneous phantom scanned using 7 transducers with the same four clinical imaging systems: a Siemens Acuson S2000, an Ultrasonix RP, a Zonare Z.one, and a VisualSonics Vevo2100. α and η estimates of lesion-mimicking spheres in the phantom were independently assessed by three research groups, who analyzed their system’s RF echo signals. Imaging-system-based estimates of α and η of both lesion-mimicking spheres were comparable to through-transmission laboratory estimates and to predictions using Faran’s theory, respectively. A few notable variations in results among the clinical systems were observed, but the average and maximum percent difference between α estimates and laboratory-assessed values was 11% and 29%, respectively. Excluding a single outlier dataset, the average and maximum average difference between η estimates for the clinical systems and values predicted from scattering theory was 16% and 33%, respectively. These results were an improvement over previous inter-laboratory comparisons of attenuation and backscatter estimates. Although the standardization of our estimation methodologies can be further improved, this study validates our results from previous rodent breast-tumor model studies.
Attenuation coefficient; backscatter coefficient; phantom; quantitative ultrasound
Three-dimensional impedance maps (3DZMs) are virtual volumes of acoustic impedance values constructed from histology to represent tissue microstructure acoustically. From the 3DZM, the ultrasonic backscattered power spectrum can be predicted and model based scatterer properties, such as effective scatterer diameter (ESD), can be estimated. Additionally, the 3DZM can be exploited to visualize and identify possible scattering sites, which may aid in the development of more effective scattering models to better represent the ultrasonic interaction with underlying tissue microstructure. In this study, 3DZMs were created from a set of human fibroadenoma samples. ESD estimates were made assuming a fluid-filled sphere form factor model from 3DZMs of volume 300 × 300 × 300 µm. For a collection of 33 independent human fibroadenoma tissue samples, the ESD was estimated to be 111 ± 40.7 µm. The 3DZMs were then investigated visually to identify possible scattering sources which conformed to the estimated model scatterer dimensions. This estimation technique allowed a better understanding of the spatial distribution and variability of the estimates throughout the volume.
Biomedical ultrasound; tissue modeling; ultra-sonic backscatter analysis; ultrasound simulation
Ultrasound (US) and ultrasound contrast agents (UCAs) provide a way to noninvasively induce targeted angiogenesis. However, there exists a lack of understanding regarding the mechanisms of this process that has impeded progress. This study sought to characterize the angiogenic response, by both exploring the role of UCA concentration ([UCA]) in bioeffect induction at 0 days post exposure (DPE) and assessing the bioeffect as a possible potentiator of angiogenesis at 5 DPE.
A 1-MHz ultrasonic transducer was used to expose the gracilis muscles of Sprague Dawley rats for 5 min with a 10-μs pulse duration, 10-Hz pulse repetition frequency, and 0.7-MPa peak rarefactional acoustic pressure (pr). Four [UCA]s were tested: 0x (saline), 1×, 5×, and 10×, where 1× is 5% Definity by volume of solution. Evans blue dye (EBD) was used to quantify changes in acute vascular permeability (0 DPE), and VEGF expression was quantified at 5 DPE to support that angiogenesis had occurred. CD31 staining was used to assess capillary density at both time points.
[UCA] was a significant parameter for determining EBD leakage (permeability) and VEGF expression (p < 0.001 for both). However, [UCA] was not a significant parameter for capillary density at 0 or 5 DPE. Multiple comparisons between 0 and 5 DPE showed that only 10× [UCA] at 5 DPE was significantly different than 0 DPE, suggesting a [UCA] dependence of the angiogenic response.
This study suggests that [UCA] was a significant parameter in the induction of an angiogenic response with US and UCAs. It also suggests that rather than damage from US and UCAs, as previously speculated, a nondestructive mechanical interaction between the UCAs and vascular endothelium induces bioeffects to potentiate the angiogenic response.
Angiogenesis; VEGF; Ultrasound-induced bioeffects; Ultrasound contrast agent; Proangiogenic therapy; Therapeutic ultrasound
TP-434 is a novel, broad-spectrum fluorocycline antibiotic with activity against bacteria expressing major antibiotic resistance mechanisms, including tetracycline-specific efflux and ribosomal protection. The mechanism of action of TP-434 was assessed using both cell-based and in vitro assays. In Escherichia coli cells expressing recombinant tetracycline resistance genes, the MIC of TP-434 (0.063 μg/ml) was unaffected by tet(M), tet(K), and tet(B) and increased to 0.25 and 4 μg/ml in the presence of tet(A) and tet(X), respectively. Tetracycline, in contrast, was significantly less potent (MIC ≥ 128 μg/ml) against E. coli cells when any of these resistance mechanisms were present. TP-434 showed potent inhibition in E. coli in vitro transcription/translation (50% inhibitory concentration [IC50] = 0.29 ± 0.09 μg/ml) and [3H]tetracycline ribosome-binding competition (IC50 = 0.22 ± 0.07 μM) assays. The antibacterial potencies of TP-434 and all other tetracycline class antibiotics tested were reduced by 4- to 16-fold, compared to that of the wild-type control strain, against Propionibacterium acnes strains carrying a 16S rRNA mutation, G1058C, a modification that changes the conformation of the primary binding site of tetracycline in the ribosome. Taken together, the findings support the idea that TP-434, like other tetracyclines, binds the ribosome and inhibits protein synthesis and that this activity is largely unaffected by the common tetracycline resistance mechanisms.
To determine whether NOX 5 is expressed in rabbit corneal stromal cells (RCSC). NADPH oxidases (NOXes) are enzymes that preferentially use NADPH as a substrate and generate superoxide. Several isoforms of NOXes function as multi-protein complexes while NOX5 and DUOXs do not require the accessory proteins for their activity and possess calcium binding EF hands.
Human NOX5 primers were used to amplify the rabbit NOX5 by RT-PCR. Amplified product was sequenced to confirm its identity. The protein encoded by the NOX5 was identified by western blot analysis. NOX5 siRNA was used to reduce transcript, protein, and calcium stimulated activity. In silico analyses were performed to establish the putative structure, functions, and evolution of rabbit NOX5.
NOX activity was measured in RCSC with NADPH rather than NADH as a substrate. RT-PCR with NOX5 primers amplified 288 bp product using RCSC cDNA, which, when sequenced, confirmed its identity to human NOX5 mRNA. This sequence was used to predict the rabbit (Oryctolagus cuniculus) NOX5 gene. NOX5 siRNA reduced amounts of NOX5 mRNA in RCSC and reduced ionomycin stimulated superoxide production. A protein of about 65 to 70 kDa encoded by the NOX5 was detected by western blot analysis. In silico analysis predicted a putative rabbit NOX5 protein containing 801 amino acids. Motif searches predicted the presence of at least 3 putative EF-hands in N-terminus and a NOX domain in C terminal region.
The data document that the NOX5 gene was expressed in cells of lagomorphs unlike rodents, making the rabbit an interesting model to study NOX5 functions. The activity of the rabbit NOX5 was calcium stimulated, a trait of NOX5 in general. NOX5 may also prove to be a useful genetic marker for studying the taxonomic position of lagomorphs and the Glires classification.
Age-dependent threshold and superthreshold behaviors of ultrasound-induced lung hemorrhage were investigated with one hundred ten 12.6±0.8-day-old rats, one hundred ten 22.9±0.8-day-old rats, and one hundred 57.7±3.9-day-old rats. Exposure conditions were: 2.8 MHz, 10-s exposure duration, 1-kHz pulse repetition frequency and 1.3-μs pulse duration. The in situ (at the pleural surface) peak rarefactional pressure (pr(insitu)) ranged between 1.4 and 10.8 MPa for which there were either 9 or 10 acoustic pressure groups for each of the three rat ages (10 rats/exposure group). For each of the three rat ages there were also shams; there were no lesions in the shams. The pr(insitu) levels were randomized within each age group; rat age was not randomized. Individuals involved in animal handling, exposure, and lesion scoring were blinded to the exposure condition. In addition, one hundred fifty six 72-day-old rats were included from three completed studies (same experimental conditions) to provide a fourth age group for the analysis. Probit regression analysis was used to examine the dependence of the occurrence of lesions on pr(insitu) in the four age groups. Likewise, lesion depth and lesion root surface area were analyzed using Gaussian tobit regression analysis. Although pr(insitu) was a significant variable, no significant age dependence of the pr(insitu) effect was found. Furthermore, age had no significant effect on either the rate of occurrence or the depth of lesions. Given the occurrence of a lesion, a weak age dependence was found for the median surface area of the induced lesion (p-value = 0.037).
lung; pulmonary hemorrhage; pulsed ultrasound; rat