Effect of Different Intensity Pulsed Ultrasound on the Restoration of Rat Skeletal Muscle Contusion

Muscle damage is a common form of injury. The incidence of muscle damage accounts for up to half of the sports injuries. The aim of this study was to investigate the effect of pulsed ultrasound on the healing process in an animal contusion injury model. SD rats (62) were randomly divided into control group (CG, 14 rats) and treatment group (48). According to the intensities of Ultrasound therapy, the treatment group was divided into 4 subgroups of 12 rats, each: A (0.25 W/cm², US₁), B (0.5 W/cm², US₂), C (0.75 W/cm², US₃) and D (0.25 W/cm²). The effectiveness of ultrasound treatment on muscle injuries was evaluated, and the optimal intensity of ultrasound in treating muscle injuries was explored. The results obtained provide experimental and theoretical evidence for the clinical effectiveness of Ultrasound therapy in treating muscle injuries.     

Ultrasound-Enhanced Drug Transport and Distribution in the Brain

Drug delivery in the brain is limited by slow drug diffusion in the brain tissue. This study tested the hypothesis that ultrasound can safely enhance the permeation of drugs in the brain. In vitro exposure to ultrasound at various frequencies (85 kHz, 174 kHz, and 1 MHz) enhanced the permeation of tritium-labeled molecules with molecular weight up to 70 kDa across porcine brain tissue. A maximum enhancement of 24-fold was observed at 85 kHz and 1,200 J/cm². In vivo exposure to 1-MHz ultrasound further demonstrated the ability of ultrasound to facilitate molecule distribution in the brain of a non-human primate. Finally, ultrasound under conditions similar to those used in vivo was shown to cause no damage to plasmid DNA, siRNA, adeno-associated virus, and fetal rat cortical neurons over a range of conditions. Altogether, these studies demonstrate that ultrasound can increase drug permeation in the brain in vitro and in vivo under conditions that did not cause detectable damage.     

Trehalose loading through the mitochondrial permeability transition pore enhances desiccation tolerance in rat liver mitochondria

Trehalose has extensively been used to improve the desiccation tolerance of mammalian cells. To test whether trehalose improves desiccation tolerance of mammalian mitochondria, we introduced trehalose into the matrix of isolated rat liver mitochondria by reversibly permeabilizing the inner membrane using the mitochondrial permeability transition pore (MPTP). Measurement of the trehalose concentration inside mitochondria using high performance liquid chromatography showed that the sugar permeated rapidly into the matrix upon opening the MPTP. The concentration of intra-matrix trehalose reached 0.29 mmol/mg protein (∼190 mM) in 5 min. Mitochondria, with and without trehalose loaded into the matrix, were desiccated in a buffer containing 0.25 M trehalose by diffusive drying. After re-hydration, the inner membrane integrity was assessed by measurement of mitochondrial membrane potential with the fluorescent probe JC-1. The results showed that following drying to similar water contents, the mitochondria loaded with trehalose had significantly higher inner membrane integrity than those without trehalose loading. These findings suggest the presence of trehalose in the mitochondrial matrix affords improved desiccation tolerance to the isolated mitochondria.     

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.     

Effect of ultrasound combined with malic acid on the activity and conformation of mushroom (Agaricus bisporus) polyphenoloxidase

Polyphenoloxidase (PPO) plays an important role in the browning of vegetables, fruits and edible fungi. The effects of ultrasound, malic acid, and their combination on the activity and conformation of mushroom (Agaricus bisporus) PPO were studied. The activity of PPO decreased gradually with the increasing of malic acid concentrations (5–60 mM). Neither medium concentrations (10, 20, 30 mM) malic acid nor individual ultrasound (25 kHz, 55.48 W/cm²) treatment could remarkably inactivate PPO. However, the inactivation during their combination was more significant than the sum of ultrasound inactivation and malic acid inactivation. The inactivation kinetics of PPO followed a first-order kinetics under the combination of ultrasound and malic acid. The conformation of combination treated PPO was changed, which was reflected in the decrease of α-helix, increase of β-sheet contents and disruption of the tertiary structure. Results of molecular microstructure showed that ultrasound broke large molecular groups of PPO into small ones. Moreover, combined treatment disrupted the microstructure of PPO and molecules were connected together.     

Changes of phosphatidylserine distribution in human red blood cells during the process of loading sugars

The plasma membrane of red blood cells permits sugars to be loaded into the cytoplasm simply by incubation in a suitable buffer solution containing the sugar. This may provide some hope for the freeze-drying of human red blood cells. However, the effect of the loading process on red blood cells has not been fully investigated. The exposure of phosphatidylserine (PS) on the surface of the cell can be recognized by macrophages and result in shortened circulation in vivo. This study evaluates the effects of the concentration, the incubation time, and the temperature of exposure of human red blood cells to extracellular trehalose or glucose. Exposure of PS was demonstrated by annexin V labeling. It was shown that the efficiency of loading of glucose was significantly greater than that of trehalose. The loading efficiency of both sugars increased with increase in extracellular sugar concentration, prolongation of incubation time, and increase of incubation temperature. The percentages of cells with exposed PS and of damaged cells were dependent on the extracellular sugar concentration, the incubation time, and the temperature. With an extracellular glucose concentration of 0.8M, the percentage of cells with exposed PS was more than 80% and significantly higher than that of red blood cells loaded with trehalose (approximate 20%, P<0.01). As the incubation time was prolonged, the percentage of PS exposure and of damaged cells also increased. After incubation for 5h, the percentage of red cells with exposed PS following loading with glucose was more than 80% and significantly higher than that of cells loaded with trehalose (40%, P<0.01). In addition, the incubation temperature had a major effect on PS exposure. The percentage of cells with PS exposure and the proportion of damaged cells increased with increase of incubation temperature. At 37 degrees C, the percentage of cells with exposed PS and of damaged cells after loading with glucose was more than 80% and significantly higher than that of cells loaded with trehalose (P<0.01). However, when the temperature was below 25 degrees C, the percentage of cells with exposed PS and of damaged cells after loading with glucose or trehalose were both less than 10%. In conclusion, the loading efficiency for glucose was higher than that for trehalose, but the lesser effect of trehalose on exposure of PS suggests that it can maintain the asymmetrical distribution of membrane phospholipids and the intracellular trehalose can increase the osmotic tolerance of cells.     

Trehalose loading through the mitochondrial permeability transition pore enhances desiccation tolerance in rat liver mitochondria

Trehalose has extensively been used to improve the desiccation tolerance of mammalian cells. To test whether trehalose improves desiccation tolerance of mammalian mitochondria, we introduced trehalose into the matrix of isolated rat liver mitochondria by reversibly permeabilizing the inner membrane using the mitochondrial permeability transition pore (MPTP). Measurement of the trehalose concentration inside mitochondria using high performance liquid chromatography showed that the sugar permeated rapidly into the matrix upon opening the MPTP. The concentration of intra-matrix trehalose reached 0.29 mmol/mg protein (approximately 190 mM) in 5 min. Mitochondria, with and without trehalose loaded into the matrix, were desiccated in a buffer containing 0.25 M trehalose by diffusive drying. After re-hydration, the inner membrane integrity was assessed by measurement of mitochondrial membrane potential with the fluorescent probe JC-1. The results showed that following drying to similar water contents, the mitochondria loaded with trehalose had significantly higher inner membrane integrity than those without trehalose loading. These findings suggest the presence of trehalose in the mitochondrial matrix affords improved desiccation tolerance to the isolated mitochondria.     

Optimization and economic evaluation of ultrasound extraction of lutein from Chlorella vulgaris

The main carotenoid in Chlorella vulgaris is lutein. The ultrasound alone or together with enzymatic pretreatment for the extraction of lutein from C. vulgaris was optimized using response surface methodology (RSM) to improve the extraction process. The optimal ultrasound extraction condition was: ultrasound frequency, 35 kHz; ultrasound intensity, 56.58 W/cm²; extraction temperature, 37.7°C; extraction time, 5 h; and ratio of solvent to solid, 31 mL/g, where the lutein recovery was 3.16 ± 0.03 mg/g wet C. vulgaris. The optimal enzymatic pretreatment was: reaction time, 2 h; enzyme concentration, 1.23% (v/w); pH, 4.5, and temperature 50°C. The optimal ultrasound extraction with enzymatic pretreatment was: ultrasound frequency, 35 kHz; ultrasound intensity, 56.58 W/cm²; extraction temperature, 37.7°C; extraction time, 162 min; and ratio of solvent to solid, 35.6 mL/g wet C. vulgaris, where the extraction yield of lutein was 3.36 ± 0.10 mg/g wet C. vulgaris. This was much higher than for ultrasound treatment alone. The surface areas of microalga cells treated by ultrasound with/without enzymatic pretreatment increased significantly, which might contribute to the increase in lutein yield. There were no significant differences in structure, color, and antioxidant activity of lutein between the ultrasound and conventional methods. The highest cost of the crude and lutein was obtained by the ultrasound with enzymatic pretreatment due to the complex process and liquid waste in the enzymatic pretreatment process, but the ultrasound treatment alone was the lowest. Therefore, ultrasound extraction is the most economical method for the extraction of microalgal lutein.     

Effects of pre-freeze incubation of human red blood cells with various sugars on postthaw recovery when using a dextran-rapid cooling protocol

Polymer has been used as substitute to replace glycerol for cryopreservation of red blood cells (RBCs). But polymer can not penetrate cell membrane, it can not efficiently protect the inner membrane. In this study, RBCs were incubated with glucose, fructose, galactose or trehalose and frozen in liquid nitrogen for 24 h using dextran as the extracellular protectant. The postthaw quality was assessed by RBC hemolysis, RBC morphology, PS distribution, osmotic fragility, and the 4 °C stability. The results indicated the loading efficiency of monosaccharide was significantly higher than that of trehalose. Adding trehalose and 40% dextran caused more serious hemolysis before freezing. The percent hemolysis of RBCs loaded with high concentration of trehalose was approximately 16% and significantly more than that of RBCs loaded with glucose (approximately 5%, P < 0.05). Intracellular trehalose can not increase the postthaw recovery of RBCs compared with cells frozen without sugar. However, low concentration of intracellular glucose or galactose can reduce the percent hemolysis to less than 5% and significantly less than that of RBCs frozen without sugar (P < 0.05). Finally, the ability of galactose or fructose to maintain the 4 °C stability was significantly more than that of glucose. In conclusion, the injuries caused by trehalose loading may directly lead to postthaw hemolysis and poor quality of RBCs. However, monosaccharide can enhance the recovery of frozen RBCs. The cryoprotective effect of galactose may be better than that of glucose or fructose. In the future, we will continue to look for a safe and efficient trehalose loading process and try to decrease the osmotic fragility of RBCs frozen with polymers and sugars.     

Advances in cryopreservation of vanilla (Vanilla planifolia Jacks.) shoot-tips: assessment of new biotechnological and cryogenic factors

The effect of dehydration, cryopreservation, and reculture conditions on growth recovery (%) of vanilla (Vanilla planifolia) shoot-tips was evaluated using a D-cryoplate procedure. Tissues were excised from in vitro grown plantlets, preconditioned on MS semisolid medium supplemented with 0.15 M trehalose for 1 d, loaded in a solution of 0.4 M sucrose or trehalose and 2 M glycerol for 30 min, and dehydrated within a laminar flow cabinet for various durations (30, 60, 90, 120, 150, and 180 min). The same preconditioning and loading treatments were compared using dehydration with vitrification solutions (PVS2 or PVS3) for 30 min at room temperature according to droplet-vitrification and V-cryoplate methods. The highest (33%) recovery of cryopreserved shoot-tips was achieved using the D-cryoplate method after 0.15 M trehalose preconditioning, loading with sucrose-glycerol solution and desiccation for 180 min. DSC analyses revealed that the osmotically active water (OAW) content of the shoot-tips was reduced from 77% (fresh weight basis) to 17% after the only effective drying duration (180 min). Melting endotherms indicated that crystallization events accompanied cryopreservation of the tissues. Proliferation of multiple shoots was obtained by indirect organogenesis. Histological analysis of the explants during post-cryopreservation recovery confirmed the organogenic nature of the callus formed after 3–4 mo of reculture in the dark on semisolid multiplication medium. This was followed by a secondary organogenesis on MS medium with kinetin (2 mg L⁻¹) and exposure to a photoperiod. At present, this is the most optimized cryopreservation protocol for shoot-tips of V. planifolia.

     

Diaphragmatic lipid peroxidation in chronically loaded rats

Recent workindicates that free radical-mediated lipid peroxidation takes placewithin the diaphragm on strenuous contraction. This phenomenon has onlybeen demonstrated using fairly artificial experimental models and hasnot been studied during the type of sustained respiratory loadingtypically seen in patients with lung disease. The purpose of thepresent study was to measure the levels of several biochemical markersof protein oxidation (protein carbonyl levels) and lipid peroxidation(8-isoprostane, reduced glutathione, and oxidized glutathione levels)in diaphragms of rats subjected to chronic respiratory loading.Respiratory loading was accomplished by tracheal banding; groups ofanimals were loaded for 4, 8, or 12 days, and a group of sham-operated unloaded animals was used as controls. After loading, animals werekilled, diaphragm contractility was assessed in vitro by using aportion of the excised diaphragm, and the remaining diaphragm and thesoleus muscles were used for biochemical analysis. We found diminishedforce generation in diaphragms from all groups of banded animalscompared with muscles from controls. For example, twitch force averaged7.8 ± 0.8 (SE) N/cm² inunloaded animals and 4.0 ± 0.4, 3.0 ± 0.4, and 3.4 ± 0.4 N/cm² in animals loaded for 4, 8, and 12 days, respectively (P < 0.0001). Loading also elicited increases in diaphragmatic proteincarbonyl concentrations (P < 0.001),and the time course of alterations in carbonyl levels paralleledloading-induced alterations in the diaphragm force-frequencyrelationship. Although loading was also associated with increases indiaphragmatic 8-isoprostane levels (P < 0.003) and reductions in diaphragm reduced glutathione levels (P < 0.003), the time course ofchanges in these latter parameters did not correspond to alterations inforce. Soleus glutathione and carbonyl levels were not altered bybanding. We speculate that respiratory loading-induced alterations indiaphragmatic force generation may be related to free radical-mediatedprotein oxidation, but not to free radical-induced lipid peroxidation.     

Intracellular trehalose via transporter TRET1 as a method to cryoprotect CHO-K1 cells

Trehalose is a promising natural cryoprotectant, but its cryoprotective effect is limited due to difficulties in transmembrane transport. Thus, expressing the trehalose transporter TRET1 on various mammalian cells may yield more trehalose applications. In this study, we ran comparative cryopreservation experiments between the TRET1-expressing CHO-K1 cells (CHO-TRET1) and the CHO-K1 cells transfected with an empty vector (CHO-vector). The experiments involve freezing under various trehalose concentrations in an extracellular medium. The freeze-thawing viabilities of CHO-TRET1 cells are higher than those of CHO-vector cells for most freezing conditions. This result differs from control experiments with a transmembrane type cryoprotectant, dimethyl sulfoxide (Me₂SO), which had similar viabilities in each condition for both cell types. We conclude that the trehalose loaded into the cells with TRET1 significantly improves the cryoprotective effect. The higher viabilities occurred when the extracellular trehalose concentration exceeded 200 mM, with 250–500 mM being optimal, and a cooling rate below 30 K/min, with 5–20 K/min being optimal.     

The Effect of High-Frequency Electric Pulses on Tumor Blood Flow In Vivo

The aim of this study was to evaluate the effect of a 5-kHz repetition frequency of electroporating electric pulses in comparison to the standard 1-Hz frequency on blood flow of invasive ductal carcinoma tumors in Balb/C mice. Electroporation was performed by the delivery of eight electric pulses of 1,000 V cm⁻¹ and 100 μs duration at a repetition frequency of 1 Hz or 5 kHz. Blood flow changes in tumors were measured by laser Doppler flowmetry. Monitoring was performed continuously for 10 min before application of the electric pulses as well as immediately after application of the electric pulses for 40 min. The delivery of electric pulses to tumors induced changes in tumor blood flow. The reduction in blood flow started after the stimulation and continued for the 40-min period of observation. There was a significant difference in blood flow changes 3 min after application of the electric pulses at 1-Hz or 5-kHz repetition frequency. However, after 3 min the difference became nonsignificant. The findings showed that the high pulse frequency (5 kHz) had an effect comparable to the 1-Hz frequency on tumor blood flow except at very short times after pulse delivery, when pulses at 5 kHz produced a more intense reduction of blood flow.     

Low-amplitude ultrasound enhances hydrodynamic-based gene delivery to rat kidney

The synergistic combination of hydrodynamic-based gene delivery and ultrasound was investigated to achieve improved gene transfer to the kidney. Plasmids encoding firefly luciferase and Erythropoietin (EPO) gene were delivered into the left kidney of rats by single or combinative application of renal vein hydrodynamic injection and ultrasound treatment with or without the addition of ultrasound contrast agents (UCA). Ultrasound exposure was found to enhance the efficiency of hydrodynamic-based gene delivery for both luciferase and EPO expression. An ultrasound exposure intensity of 2 W/cm² at 10% duty cycle for 15 min, produced a maximal gene expression 4.5 times higher than hydrodynamic delivery alone. Duration, location, and tissue-specificity of gene expression were not changed by ultrasound exposure. Application of UCA reduced the intensity and exposure duration of ultrasound treatment needed for optimal expression. Appropriate application of ultrasound and UCA did not alter histological structure or impair physiological function of the treated kidney.     

The survival of amphibian embryos after continuous ultrasonic treatment

The effect of continuous ultrasonic treatment on the development of early embryos of common frog Rana temporaria was studied. Intact embryos at the blastula stage were exposed to ultrasound of various frequencies (0.88 and 2.64 MHz) and intensities (0.05 to 1.0 W/cm²) for various periods (1 to 15 min). The increase in ultrasound intensity to 0.7–1.0 W/cm² and exposure time to 5–15 min resulted in nearly complete mortality. Development of the embryos exposed to ultrasound of medium intensity (0.2–0.7 W/cm²) for 1–5 min was virtually similar to the control. Treatment at a frequency of 2.64 MHz and intensity of 0.05–0.7 W/cm² for 1–5 min had no effect on the development of amphibian embryos and their survival rate. The increase in intensity of the ultrasound of this frequency to 1 W/cm² and the exposure time to 5 min decreased the number of normally developing embryos by 35%.     

Biochemical Changes in Rat Brain Exposed to Low Intensity 9.9 GHz Microwave Radiation

Present study concerns with various biochemical changes in the developing rat brain exposed to 9.9 GHz (square wave modulated, 1 kHz) at power density 0.125 mW/cm² (specific absorption rate 1.0 W/kg) for 2 h/day for 35 days. Thirty days old male wistar rats were used for this present study. Each group consists of eight animals. After the exposure, biochemical assays such as calcium ion efflux, calcium-dependent protein kinase (PKC), and ornithine decarboxylase (ODC) were performed on the brain tissue. Results of this study reveal that chronic exposure of rat to microwave radiation alter the activity of certain enzymes. There was a significant increase in calcium ion efflux and the activity of ODC. On the other hand, there is a significant decrease in PKC activity. Since these enzymes are related to growth, any alteration may lead to affect functioning of the brain and its development.     

Effect of Ultrasound on Chromosomes of Lymphocyte Cultures

In-vitro experiments have been undertaken to assess the effect of ultrasound at diagnostic and therapeutic levels on chromosomes of human lymphocyte cultures. The frequency used was about 2 MHz. The intensity was that of diagnostic level, 23 mW/cm², which was increased stepwise to 3·5 W/cm². The time of exposure varied from two to eight hours. No difference could be detected between the numbers of cells with aberrations in the insonated and control cultures, but aggregation of red cells was observed at high intensities.     

Sub-0.6 eV Inverted Metamorphic GaInAs Cells Grown on Inp and GaAs Substrates for Thermophotovoltaics and Laser Power Conversion

Inverted metamorphic Ga_0.3In_0.7As photovoltaic converters with sub-0.60 eV bandgaps grown on InP and GaAs are presented. Threading dislocation densities are 1.3 ± 0.6 × 10⁶ and 8.9 ± 1.7 × 10⁶ cm⁻² on InP and GaAs, respectively. The devices generate open-circuit voltages of 0.386 and 0.383 V, respectively, under irradiance producing a short-circuit current density of ≈10 A cm⁻², yielding bandgap-voltage offsets of 0.20 and 0.21 V. Power and broadband reflectance measurements are used  to estimate thermophotovoltaic (TPV) efficiency. The InP-based cell is estimated to yield 1.09 W cm⁻² at 1100 °C versus 0.92 W cm⁻² for the GaAs-based cell, with efficiencies of 16.8 versus 9.2%. The efficiencies of both devices are limited by sub-bandgap absorption, with power weighted sub-bandgap reflectances of 81% and 58%, respectively, the majority of which is assumed to occur in the graded buffers. The 1100 °C TPV efficiencies are estimated to increase to 24.0% and 20.7% in structures with the graded buffer removed, if previously demonstrated reflectance is achieved. These devices also have application to laser power conversion in the 2.0–2.3 µm atmospheric window. Peak laser power converter efficiencies of 36.8% and 32.5% are estimated under 2.0 µm irradiances of 1.86 and 2.81 W cm⁻², respectively.     

Enhanced saccharification of wheat straw with the application of ultrasonic-assisted quaternary ammonium hydroxide pretreatment

Lignocelluloses featuring complicated structure and poor degradability usually require pretreatment before its utilization. In this study, an ultrasonic-assisted pretreatment by using quaternary ammonium hydroxide was introduced to enhance biodegradability of lignocellulosic biomass. The synergistic chemical and mechanical pretreatment were supposed to be responsible for both external surface destruction and internal structure disruption of lignocelluloses. High-efficient lignin removal accompanied with obvious structural (crystallinity) transformation was achieved in the pretreated straws. Process analysis indicated that factors of time, temperature, concentration of solvent, and ultrasound power intensity turned out to be significant for pretreatment, and a 4-fold increased saccharification yield of around 92.4% as compared to untreated straw was obtained from the wheat straw pretreated by 15% solvent at 50 °C for 0.5 h in power intensity 344 W/cm². All results suggest that the combined chemical and mechanical treatment can significantly improve the bio-accessibility of lignocelluloses, leading to the enhanced utilization efficiency.     

Cocultivation of avian orthomyxoviruses and paramyxoviruses in embryonated eggs: implications for surveillance studies.

A relatively inexpensive flow microcalorimeter is described which is capable of detecting heat outputs as low as 3 μW (precision, ±2%). Its use is illustrated on river epilithon (0.8 to 6.8 μW cm⁻²), river sand (9.8 μW cm⁻³), and marine sand (15.3 μW cm⁻³); however, it could be used to detect the heat output from any biotic material over which a flow of water can be passed, provided that such an action would not be disruptive to chemical and biological equilibria.