There is now a relatively large body of evidence suggesting a relationship between dysfunction of myelin and oligodendrocytes and the etiology of several neuropsychiatric disorders, including depression and schizophrenia, and also suggesting that ultrasound methods may alleviate some of the symptoms of depression. We have applied low-intensity pulsed ultrasound (LIPUS) to the brains of mice treated with the demyelinating drug cuprizone, a drug that has been used as the basis for a rodent model relevant to a number of psychiatric and neurologic disorders including depression, schizophrenia, and multiple sclerosis. Prior to conducting the studies in mice, preliminary studies were carried out on the effects of LIPUS in vitro in neuron-like SH-SY5Y cells and primary glial cells. In subsequent studies in mice, female C57BL/6 mice were restrained in plastic tubes for 20 min daily with the ultrasound transducer near the end of the tube directly above the mouse's head. LIPUS was used at an intensity of 25 mW/cm(2) once daily for 22 days in control mice and in mice undergoing daily repetitive restraint stress (RRS). Behavioral or neurochemical studies were done on the mice or the brain tissue obtained from them. The studies in vitro indicated that LIPUS stimulation at an intensity of 15 mW/cm(2) delivered for 5 min daily for 3 days in an enclosed sterile cell culture plate in an incubator increased the viability of SH-SY5Y and primary glial cells. In the studies in mice, LIPUS elevated levels of doublecortin, a marker for neurogenesis, in the cortex compared to levels in the RRS mice and caused a trend in elevation of brain levels of brain-derived neurotrophic factor in the hippocampus relative to control levels. LIPUS also increased sucrose preference (a measure of the attenuation of anhedonia, a common symptom of several psychiatric disorders) in the RRS model in mice. The ability of LIPUS administered daily to rescue damaged myelin and oligodendrocytes was studied in mice treated chronically with cuprizone for 35 days. LIPUS increased cortex and corpus callosum levels of myelin basic protein, a protein marker for mature oligodendrocytes, and neural/glial antigen 2, a protein marker for oligodendrocyte precursor cells, relative to levels in the cuprizone + sham animals. These results of this exploratory study suggest that future comprehensive time-related studies with LIPUS on brain chemistry and behavior related to neuropsychiatric disorders are warranted.

Schizochytrium sp. is a microalga that is known for its high content of oils or lipids. It has a high percentage of polyunsaturated fatty acids in the accumulated oil, especially docosahexaenoic acid (DHA). DHA is an important additive for the human diet. Large-scale production of Schizochytrium sp. can serve as an alternative source of DHA for humans as well as for fish feed, decreasing the burden on aqua systems. Therefore, research on improving the productivity of Schizochytrium attracts a lot of attention. We studied the potential of using low-intensity pulsed ultrasound (LIPUS) in the growth cycle of Schizochytrium sp. in shake flasks. Different intensities and treatment durations were tested. A positive effect of LIPUS on biomass accumulation was observed in the Schizochytrium sp. culture. Specifically, LIPUS stimulation at the ultrasound intensity of 400 mW/cm2 with 20 min per treatment 10 times a day with equal intervals of 2.4 h between the treatments was found to enhance the growth of Schizochytrium biomass most effectively (by up to 20%). Due to the nature of cell division in Schizochytrium sp. which occurs via zoospore formation, LIPUS stimulation was inefficient if applied continuously during all 5 days of the growth cycle. Using microscopy, we studied the interval between zoospore formation in the culture and selected the optimal LIPUS application days (Days 0-1 and Days 4-5 of the 5-day growth cycle). Microscopic images have also shown that LIPUS stimulation enhances zoospore formation in Schizochytrium sp., leading to more active cell division in the culture. This study shows that LIPUS can serve as an additional tool for cost-efficiency improvement in the large-scale production of Schizochytrium as a sustainable and environmentally friendly source of omega-3 (DHA).

Generating renewable energy while sequestering CO 2 using algae has recently attracted significant research attention, mostly directing towards biological methods such as systems biology, genetic engineering and bio-refining for optimizing algae strains. Other approaches focus on chemical screening to adjust culture conditions or culture media. We report for the first time the physiological changes of algal cells in response to a novel form of mechanical stimulation, or a pulsed wave at the frequency of 1.5 MHz and the duty cycle of 20%. We studied how the pulsed wave can further increase algal lipid production on top of existing biological and chemical methods. Two commonly used algal strains, fresh-water Chlorella vulgaris and seawater Tetraselmis chuii , were selected. We have performed the tests in shake flasks and 1 L spinner-flask bioreactors. Conventional Gravimetric measurements show that up to 20% increase for algal lipid could be achieved after 8 days of stimulation. The total electricity cost needed for the stimulations in a one-liter bioreactor is only one-tenth of a US penny. Gas liquid chromatography shows that the fatty acid composition remains unchanged after pulsed-wave stimulation. Scanning electron microscope results also suggest that pulsed wave stimulation induces shear stress and thus increases algal lipid production.

Vaccination is a safe and effective approach to prevent deadly diseases. To increase vaccine production, we propose that a mechanical stimulation can enhance protein production. In order to prove this hypothesis, Sf9 insect cells were used to evaluate the increase in the expression of a fusion protein from hepatitis B virus (HBV S1/S2). We discovered that the ultrasound stimulation at a frequency of 1.5 MHz, intensity of 60 mW/cm2, for a duration of 10 minutes per day increased HBV S1/S2 by 27%. We further derived a model for transport through a cell membrane under the effect of ultrasound waves, tested the key assumptions of the model through a molecular dynamics simulation package, NAMD (Nanoscale Molecular Dynamics program) and utilized CHARMM force field in a steered molecular dynamics environment. The results show that ultrasound waves can increase cell permeability, which, in turn, can enhance nutrient / waste exchange thus leading to enhanced vaccine production. This finding is very meaningful in either shortening vaccine production time, or increasing the yield of proteins for use as vaccines.

In therapeutic ultrasound applications, accurate ultrasound output intensities are crucial because the physiological effects of therapeutic ultrasound are very sensitive to the intensity and duration of these applications. Although radiation force balance is a benchmark technique for measuring ultrasound intensity and power, it is costly, difficult to operate, and compromised by noise vibration. To overcome these limitations, the development of a low-cost, easy to operate, and vibration-resistant alternative device is necessary for rapid ultrasound intensity measurement. Therefore, we proposed and validated a novel two-layer thermoacoustic sensor using an artificial neural network technique to accurately measure low ultrasound intensities between 30 and 120 mW/cm2. The first layer of the sensor design is a cylindrical absorber made of plexiglass, followed by a second layer composed of polyurethane rubber with a high attenuation coefficient to absorb extra ultrasound energy. The sensor determined ultrasound intensities according to a temperature elevation induced by heat converted from incident acoustic energy. Compared with our previous one-layer sensor design, the new two-layer sensor enhanced the ultrasound absorption efficiency to provide more rapid and reliable measurements. Using a three-dimensional model in the K-wave toolbox, our simulation of the ultrasound propagation process demonstrated that the two-layer design is more efficient than the single layer design. We also integrated an artificial neural network algorithm to compensate for the large measurement offset. After obtaining multiple parameters of the sensor characteristics through calibration, the artificial neural network is built to correct temperature drifts and increase the reliability of our thermoacoustic measurements through iterative training about ten seconds. The performance of the artificial neural network method was validated through a series of experiments. Compared to our previous design, the new design reduced sensing time from 20 s to 12 s, and the sensor’s average error from 3.97 mW/cm2 to 1.31 mW/cm2 respectively.

Hepatitis B is an infectious liver disease and vaccination is an effective way to protect individuals. We have applied mechanical wave stimulation to increase protein production. To validate our design, we used Sf9 insect cells to increase antigen fragment fusion protein expression for hepatitis B virus (HBV S1/S2). We discovered that stimulation at a frequency of 1.5MHz, intensity of 60mW/ cm(2), for a duration of 10 minutes per day increased HBV S1/S2 production by 15%. This finding is very significant for shortening vaccine production time or increasing the yield of proteins for use as vaccines.

Generating renewable energy while sequestering CO 2 using algae has recently attracted significant research attention, mostly directed towards biological methods such as systems biology, genetic engineering and bio-refining for optimizing algae strains. Other approaches focus on chemical screening to adjust culture conditions or culture media. We report for the first time the physiological changes of algal cells in response to a novel form of mechanical stimulation, or a pulsed wave at the frequency of 1.5MHz and the duty cycle of 20%. Most importantly, we studied how the pulsed wave can further increase algal oil production on top of existing biological and chemical methods. Two commonly used algal strains, fresh-water Chlorella vulgaris and seawater Tetraselmis chuii, were selected. Conventional Gravimetric measurements show that up to 20% increase for T. chuii at the intensity of 80 mW/cm 2 and 10% increase for C. vulgaris in algal oil could be achieved after 8 days of stimulation at the intensity of 100 mW/cm 2 . Gas chromatography / mass spectrometry (GC/MS) shows that the fatty acid composition remains unchanged after pulsed-wave stimulation. Scanning electron microscope results also suggest that pulsed wave stimulation induces shear stress and thus increase algal oil production.

Many technologies, such as cell line screening and host cell engineering, culture media optimization and bioprocess optimization, have been proposed to increase monoclonal antibody (mAb) production in Chinese Hamster Ovary (CHO) cells. Unlike the existing biochemical approaches, we investigated stimulation using low-intensity pulsed ultrasound (LIPUS) as a purely physical approach, offering enhanced scalability, contamination control and cost-efficiency, while demonstrating significantly increased cell growth and antibody production. It was found that daily ultrasound treatments at 40 mW/cm2 for 5 min during cell culture increased the production of human anti-IL-8 antibody by more than 30% using 10 or 30 mL shake flasks. Further increasing the ultrasound dosage (either intensities or the treatment duration) did not appreciably increase cell growth or antibody production, however feeding the culture with additional highly-concentrated nutrients, glucose and amino acids (glutamine in this case), did further increase cell growth and antibody titer to 35%. Similar ultrasound treatments (40 mW/cm2, 5 min per day) when scaled up to larger volume wavebags, resulted in a 25% increase in antibody production. Increased antibody production can be attributed to both elevated cell count and the ultrasound stimulation. Theoretical study of underlying mechanisms was performed through the simulations of molecular dynamics using the AMBER software package, with results showing that LIPUS increases cell permeability. The significance of this study is that LIPUS, as a physical-based stimulation approach, can be externally applied to the cell culture without worrying about contamination. By combining with the existing technologies in antibody production, LIPUS can achieve additional mAb yields. Because it can be easily integrated with existing cell culture apparatuses, the technology is expected to be more acceptable by the end users.

In this paper, a simple and adaptive thermoacoustic sensor was designed to measure Low Intensity Pulsed Ultrasound (LIPUS). Compared to other thermoacoustic sensor designs, our novelty lies in (i) integrating an ultrasound medium layer during the measurement to simplify the complicated set-up procedures and (ii) taking the effect of ambient temperatures into design consideration. After measuring temperature increases with various ambient temperatures under different ultrasound intensities, a relationship among ultrasound intensities, ambient temperatures and coefficients of temporal temperature changes was calculated. Our improved design has made the sensor easy to operate and its performance more accurate and consistent than the thermoacoustic sensor designs without considering ambient temperatures. In all, our improved design greatly enhances the thermoacoustic sensor in practical ultrasound calibration.

We explored the application of Low-Intensity Pulsed Ultrasound (LIPUS) technology to improve the metabolic activity of microorganisms. In this study we showed that LIPUS improves bio-ethanol production from lignocellulosic biomass. We determined specific LIPUS conditions to increase the metabolic activity of both the cellulose degrading fungus, Trichoderma reesei Rut C-30 and the ethanol producing yeast, Saccharomyces cerevisiae. LIPUS conditions of 1.5 MHz, 20% duty cycle, 80 mW/cm(2) intensity, 5 min exposure and 12 exposures per day were found to improve the activity of the organisms the most. These LIPUS treatment conditions increased cellulase production by T reesei by 16 +/- 6%. The same LIPUS treatment conditions induced a 31 +/- 10% increase in ethanol production by S. cerevisiae which implies a cumulative improvement of 52 +/- 16% in lignocellulosic bio-ethanol production with LIPUS. This observation shows a new potential for LIPUS in the improvement of lignocellulosic bioethanol production to make it a sustainable energy source. (C) 2013 Elsevier Ltd. All rights reserved.