Free Radical Formation by Ultrasound in Aqueous Solutions. A Spin Trapping Study

Our recent spin trapping studies of free radical generation by ultrasound in aqueous solutions are reviewed. The very high temperatures and pressures induced by acoustic cavitation in collapsing gas bubbles in aqueous solutions exposed to ultrasound lead to the thermal dissociation of water vapor into H atoms and OH radicals. Their formation has been confirmed by spin trapping. Sonochemical reactions occur in the gas phase (pyrolysis reactions), in the gas-liquid interfacial region, and in the bulk of the solution (radiation-chemistry reactions). The high temperature gradients in the interfacial regions lead to pyrolysis products from non-volatile solutes present at sufficiently high concentrations. The sonochemically generated radicals from carboxylic acids, amino acids, dipeptides. sugars, pyrimidine bases. nucleosides and nucleo-tides were identified by spin trapping with the non-volatile spin trap 3.5-dibromo-2.6-dideuterio-4-nitrosobenzenesulfonate. At low concentrations of the non-volatile solutes. the spin-trapped radicals produced by sonolysis are due to H atom and OH radical reactions. At higher concentrations of these non-volatile solutes, sonolysis leads to the formation of additional radicals due to pyrolysis processes (typically methyl radicals). A preferred localization of non-volatile surfactants (compared to analogous non-surfactant solutes) was demonstrated by the detection of pyrolysis radicals at 500-fold lower concentrations. Pyrolysis radicals were also found in the sonolysis of aqueous solutions containing only certain nitrone spin traps. The more hydrophobic the spin trap, the lower the concentration at which the pyrolysis radicals can be observed. The effect of varying the temperature of collapsing transient cavities in aqueous solutions of different rare gases and of N₂O on radical yields and on cell lysis of mammalian cells was investigated.     

Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimate viability by means of vital dyes. Methylene blue, Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans.Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium.In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25 %. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.     

Comparison of ultrasound and skinfold measurements in assessment of subcutaneous and total fatness

Ultrasound (A-scan mode) and skinfold methods were evaluated in the measurement of subcutaneous fat thickness and prediction of total fat weight (by whole body potassium counting). Based on intraobserver correlations on 39 men at 15 body sites, skinfold caliper measurements were more reproducible than ones obtained by ultrasound. Measurements made with the two techniques at the same site typically produced different mean estimates of fat thickness. However, scores were often highly correlated with each other, indicating similar relative rankings of subjects by each technique. Skinfolds were more highly correlated with total fat weight than were ultrasound measurements, but body weight and anthropometric measures had even higher correlations with total fat weight. Anthropometric measurements were highly correlated with fatness because of their association with body weight, and when this relationship was statistically controlled for, they typically lost their predictive effectiveness. Multiple regression analyses revealed that the best predictors of fat weight were body weight along with skinfold and ultrasound measurements. These results suggest that skinfolds are a more effective means of assessing subcutaneous fat than ultrasound, especially when the large difference in cost of equipment is considered.     

Ultrasound effect on physical properties of corn starch

High power ultrasound (HPU) represents a non-thermal processing method that has been rapidly researched and used in the last 10 years. The application of power ultrasound offers the opportunity to modify and improve some technologically important compounds which are often used in food products. One of them is starch. The aim of this research was to examine the effect of the high power ultrasound of 24 kHz frequency on rheological and some physical properties of corn starch. Various ultrasound treatments were used; an ultrasound probe set with different intensities (34, 55, 73 W cm⁻²) and treatment times (15 and 30 min) and ultrasound bath of 2 W cm⁻² intensity and treatment times (15 and 30 min). Rheological parameters, turbidity and swelling power of corn starch suspensions were determined for native and ultrasonically treated corn starch suspensions. Differential scanning calorimetry was used in order to examine the pasting properties of corn starch. The results have shown that the ultrasound treatment of corn starch distorts the crystalline region in starch granules. The results of differential scanning calorimetry measurements have shown a decrease in enthalpy of gelatinization. A significant decrease in consistency coefficient (k) has also been observed. The consistency coefficient decreases stepwise jointly with the increasing ultrasound power. The increase in the swelling power is associated with water absorption capacity and corn starch granules solubility, respectively.     

Emerging technologies in therapeutic ultrasound: Thermal ablation to gene delivery

Ultrasound is used today in medicine as a modality for diagnostic imaging. Recently, there have been numerous reports on the application of thermal and nonthermal ultrasound energy for treating various diseases. In addition to thermal ablation of tumors, non-thermal ultrasound combined with drugs and genes have led to much excitement especially for cancer treatment, vascular diseases, and regenerative medicine. Ultrasound energy can enhance the effects of thrombolytic agents such as urokinase for treatment of stroke and acute myocardial infarction. New ultrasound technologies have resulted in advanced devices such as a) ultrasound catheters, b) Non-invasive methods as high intensity focused ultrasound (HIFU) in conjunction with MRI and CT is already being applied in the clinical field, c) Chemical activation of drugs by ultrasound energy for treatment of tumors is another new field recently termed "Sonodynamic Therapy", and d) Combination of genes and microbubble have induced great hopes for ideal gene therapy (sonoporation). Various examples of ultrasound combined modalities are under investigation which could lead to revolutionary therapy.     

Ultrasound-assisted liquid-state fermentation of soybean meal with Bacillus subtilis: Effects on peptides content,ACE inhibitory activity and biomass

This work studied the effects of ultrasound-assisted liquid-state fermentation on the content of peptides and bioactivity of fermented soybean meal (FSBM) with Bacillus subtilis. Based on the optimization of basic ultrasonic parameters, effects of the main parameters including power density, incubation temperature and ultrasonic time on in vitro ACE inhibitory activity, peptides content and biomass of B. subtilis were investigated via Box-Behnken experimental design. Three mathematical models were determined to show the effects of each variable and their combinatorial interactions on dependent variables. A set of preferred condition was determined to be at power density of 0.08 W/mL for 1 h with an initial incubation temperature of 36.7 °C, according to the desirability function analyses. Under these conditions, the experimental values of in vitro ACE inhibitory activity, peptides content and biomass increased by 26.4%, 36.2% and 55.0% compared to those of the control, respectively, accompanied by the improvement of the peptides yield, purity and conversion rate in varying degrees. Additionally, FSBM resulted in a significant decrease of systolic blood pressure by 20.7 ± 1.8 mmHg in spontaneously hypertensive rats at 3 h. These results suggest that ultrasonication is an efficient solution for enhancing fermentation to produce bioactive components from agroindustrial by-products.     

Ultrasound Enhances Retrovirus‐Mediated Gene Transfer

Abstract

Viral vector systems are efficient for transfection of foreign genes into many tissues. Especially, retrovirus based vectors integrate the transgene into the genome of the target cells, which can sustain long term expression. However, it has been demonstrated that the transduction efficiency using retrovirus is relatively lower than those of other viruses. Ultrasound was recently reported to increase gene expression using plasmid DNA, with or without, a delivery vehicle. However, there are no reports, which show an ultrasound effect to retrovirus‐mediated gene transfer efficiency.

Retrovirus‐mediated gene transfer systems were used for transfection of 293T cells, bovine aortic endothelial cells (BAECs), rat aortic smooth muscle cells (RASMCs), and rat skeletal muscle myoblasts (L6 cells) with β‐galactosidase (β‐Gal) genes. Transduction efficiency and cell viability assay were performed on 293T cells that were exposed to varying durations (5 to 30 seconds) and power levels (1.0 watts/cm² to 4.0 watts/cm²) of ultrasound after being transduced by a retrovirus. Effects of ultrasound to the retrovirus itself was evaluated by transduction efficiency of 293T cells. After exposure to varying power levels of ultrasound to a retrovirus for 5 seconds, 293T cells were transduced by a retrovirus, and transduction efficiency was evaluated.

Below 1.0 watts/cm² and 5 seconds exposure, ultrasound showed increased transduction efficiency and no cytotoxicity to 293T cells transduced by a retrovirus. Also, ultrasound showed no toxicity to the virus itself at the same condition. Exposure of 5 seconds at the power of 1.0 watts/cm² of an ultrasound resulted in significant increases in retrovirus‐mediated gene expression in all four cell types tested in this experiment. Transduction efficiencies by ultrasound were enhanced 6.6‐fold, 4.8‐fold, 2.3‐fold, and 3.2‐fold in 293T cells, BAECs, RASMCs, and L6 cells, respectively. Furthermore, β‐Gal activities were also increased by the retrovirus with ultrasound exposure in these cells.

Adjunctive ultrasound exposure was associated with enhanced retrovirus‐mediated transgene expression in vitro. Ultrasound associated local gene therapy has potential for not only plasmid‐DNA‐, but also retrovirus‐mediated gene transfer.     

Acoustic sensing and signal processing techniques for monitoring milk fouling cleaning operations

Large resource investments are necessary in order to minimize the limiting problems arising from food industrial intensive productivity. One of the most challenging concerns is the cleaning status uncertainty among heat transfer areas in dairy heat exchangers, since the effectiveness of this process cannot be easily validated. The present study aimed to develop a low‐power ultrasound sensing method for monitoring the removal of milk fouling deposits along cleaning processes inside an experimental plate heat exchanger structure, connected to a milk piping unit. For that purpose, signal processing, namely acoustic feature extraction, over different wave patterns combined with artificial neural network techniques was used. Measurements were taken in pulse‐echo mode with a handmade 4 MHz ultrasound transducer. While fouling deposits having initial average thickness values of 250 μm (34.5 ± 4.5 mg/cm²) were removed, the acoustic transmissivity increased. Results showed that the signal features follow the expected trends in both, clean and fouled cases, within right guess detection accuracies above 80%. Therefore, when calibrated well, this could be a very sensitive and noninvasive technique for material characterization, as well as a suitable validation method for industrial cleaning cycle operation optimization that could significantly reduce the associated costs.     

BI-RADS 3级对乳腺病灶患者的诊断结果及影响因素分析

目的:回顾性分析超声BI-RADS 3级对乳腺病灶患者的诊断结果及影响因素。方法:选择2014年8月至2017年8月上海交通大学医学院附属第九人民医院和复旦大学附属华山医院北院收治的168例乳腺病灶患者,回顾性分析其影像学资料及病理分析结果。分析BI-RADS 3级对乳腺病灶的阴性诊断率,采用Kim分级对BI-RADS 3级结果进行重新分级,分析影响BI-RADS分级及重新分级的主要因素。结果:168例BI-RADS 3级乳腺病灶中,159例为良性病变,9例为恶性。168例BI-RADS 3级乳腺病灶的阳性预测值为5.4%(9/168),阴性预测值为94.6%(159/168)。病灶数目、年龄、医师年资、病灶大小对BI-RADS 3级良恶性乳腺病灶判断无明显影响,而BI-RADS 3级恶性乳腺病灶较良性病灶更易触诊(P 0.05)。重新分级发现,124例仍为BI-RADS 3级,44例上升至BI-RADS 4级,重新分级恶性病灶的检出率为100%(9/9),假阳性率为20.8%(35/168)。病灶多发、年龄≥40岁更可能评估为BI-RADS 4级(P0.05),医师年资、病灶大小、病灶触及情况对重新分级无明显影响(P0.05)。结论:BI-RADS 3级对乳腺良性病灶有较高的阴性诊断率,重新分级可提高乳腺恶性病灶的检出率,但会造成较高的假阳性率,影响BI-RADS分级的主要因素为病灶可否扪及,影响重新分级的主要因素为患者年龄及乳腺病灶是否多发。     

The shear modulus of lower-leg muscles correlates to intramuscular pressure

Shear wave elastography (SWE) is emerging as an innovative tool to evaluate muscle properties and function. It has been shown to correlate with both passive and active muscle forces, and is sensitive to physiological processes and pathological conditions. Similarly, intramuscular pressure (IMP) is an important parameter that changes with passive and active muscle contraction, body position, exercise, blood pressure, and several pathologies. Therefore, the objective of this study was to quantify the dependency of shear modulus within the lower-leg muscles on IMP in healthy individuals. Nineteen healthy individuals (age: Mean age ± SD, 23.84 ± 6.64 years) were recruited. Shear modulus was measured using ultrasound SWE on the tibialis anterior (TA) and peroneus longus (PL) muscles using pressure cuff inflation around the thigh at 40 mmHg, 80 mmHg, and 120 mmHg. Changes in IMP were verified using a catheter connected to a blood pressure monitor. It was found that IMP was correlated to TA and PL shear modulus (spearman's rank correlation = 0.99 and 0.99, respectively). Applying a gradual increase of cuff pressure from 0 to 120 mmHg increased the shear modulus of the TA and PL muscles from 15.83 (2.46) kPa to 21.88 (4.33) kPa and from 9.64 (1.97) kPa to 12.88 (5.99) kPa, respectively. These results demonstrate that changes of muscle mechanical properties are dependent on IMP. This observation is important to improve interpretation of ultrasound elastograms and to potentially use it as a biomarker for more accurate diagnosis of pathologies related to increased IMP.