Citations

ABSTRACT Measles virus (MV) lacking expression of C protein (C KO ) is a potent activator of the double-stranded RNA (dsRNA)-dependent protein kinase (PKR), whereas the isogenic parental virus expressing C protein is not. Here, we demonstrate that significant amounts of dsRNA accumulate during C KO mutant infection but not following parental virus infection. dsRNA accumulated during late stages of infection and localized with virus replication sites containing N and P proteins. PKR autophosphorylation and stress granule formation correlated with the timing of dsRNA appearance. Phospho-PKR localized to dsRNA-containing structures as revealed by immunofluorescence. Production of dsRNA was sensitive to cycloheximide but resistant to actinomycin D, suggesting that dsRNA is a viral product. Quantitative PCR (qPCR) analyses revealed reduced viral RNA synthesis and a steepened transcription gradient in C KO virus-infected cells compared to those in parental virus-infected cells. The observed alterations were further reflected in lower viral protein expression levels and reduced C KO virus infectious yield. RNA deep sequencing confirmed the viral RNA expression profile differences seen by qPCR between C KO mutant and parental viruses. After one subsequent passage of the C KO virus, defective interfering RNA (DI-RNA) with a duplex structure was obtained that was not seen with the parental virus. We conclude that in the absence of C protein, the amount of PKR activator RNA, including DI-RNA, is increased, thereby triggering innate immune responses leading to impaired MV growth.

Abstract Ovarian cancer is a clinically and molecularly heterogeneous disease. The driving forces behind this variability are unknown. Here, we report wide variation in the expression of the DNA cytosine deaminase APOBEC3B, with elevated expression in the majority of ovarian cancer cell lines (three SDs above the mean of normal ovarian surface epithelial cells) and high-grade primary ovarian cancers. APOBEC3B is active in the nucleus of several ovarian cancer cell lines and elicits a biochemical preference for deamination of cytosines in 5′-TC dinucleotides. Importantly, examination of whole-genome sequence from 16 ovarian cancers reveals that APOBEC3B expression correlates with total mutation load as well as elevated levels of transversion mutations. In particular, high APOBEC3B expression correlates with C-to-A and C-to-G transversion mutations within 5′-TC dinucleotide motifs in early-stage high-grade serous ovarian cancer genomes, suggesting that APOBEC3B-catalyzed genomic uracil lesions are further processed by downstream DNA "repair" enzymes including error-prone translesion polymerases. These data identify a potential role for APOBEC3B in serous ovarian cancer genomic instability. Cancer Res; 73(24); 7222–31. ©2013 AACR.

Vibro-acoustography is based on audio-frequency harmonic vibrations induced in the object by the radiation force of focused ultrasound. The resulting object vibration produces a hydrophone detectable acoustic emission that is a function of regional dynamic properties of the object at the vibration frequency. The amplitude of the hydrophone signal is mapped into an image that represents the object’s vibro-acoustic response to the radiation force by scanning the focused ultrasound across the object. Contrast in Vibro-acoustography images represent several properties of the object, including the dynamic characteristics at the audio frequency and scattering and absorption properties at the ultrasound frequency. A Vibro-acoustography system has been combined with a stereo-tactic x-ray mammography system for in vivo breast imaging and has been tested on a number of volunteers. Resulting images show soft tissue structures and microcalcifications within breast with high contrast, high resolution, and no speckles. The results have been verified using x-ray mammograms of the breast. The encouraging results from in vitro and in vivo experiments suggest further development of vibro-acoustography may lead to a new clinical breast imaging modality. [Work supported in part by a grant from the Susan G. Komen Breast Cancer Foundation. Vibro-acoustography is patented by MF and JFG.]

The fact that modulated ultrasound at a pulse intensity of 160 mW/cm2 can induce fracture healing implies that cells that are sensitive to strain can feel and respond to this signal. A hypothesis that uses Nyborg inspired methods to investigate this effect will be presented along with data from a wide variety of simulations and experimental data. It appears that the response is mediated through the cytoskeleton and may be associated with interaction of the sound with focal adhesion complexes at one end of the skeletal fibers for high frequency responses and with the nuclear membrane at the other end of the fibers for low frequency responses. Although this hypothesis is nascent it is supported with experimental data from the literature.

In vitro studies have shown that treatment with 1.5 MHz ultrasound signal (160 mW/cm2) at a 200 μs tone burst repeating at 1 kHz increases proteoglycan synthesis in chondrocytes [J. Parvisi et al., J. Orthop. Res. 17, 488–494 (1999)]. It was hypothesized that a continuous 1 kHz signal would be similar to the pulsed 1.5 MHz signal in stimulating chondrocytes to produce proteoglycan, which may cause accelerated fracture healing. In vitro experiments were performed with ATDC5 cells, a chondrogenic clonal cell line, plated in 6-well plates for 3 to 7 days before receiving ultrasound treatments. Cells were treated with either 1.5 MHz pulsed signal or 1 kHz signal for 20 minutes per day for 9 to 11 days. The signals were calibrated so that the bottom of the 6-well plate moved 10 nm for each condition. After the final treatment, cell layers were stained with Alcian blue, which stains cartilage nodules providing a measure of chondrogenesis. Both 1.5 MHz and 1 kHz led to a highly significant increase in chondrogenesis compared to control. Quantitative image analysis of stained wells showed that treatments with either signal increased number of nodules 2.3-fold (p<0.02) and total area of nodules 3-fold (p<0.02) compared to controls.

With the advent of MEMS, modal analysis of small structures is increasingly important. However, conventional excitation techniques normally require contact, which may not be feasible for small objects. We present a non-contact method that uses interference of ultrasound frequencies in air to produce low-frequency excitation of structures. Objects studied included hard-drive HGA suspensions and MEMS devices. The vibration induced by the ultrasound radiation force was varied in a wide range from 0 Hz to 50 kHz. Object motion was detected using a laser vibrometer; measured frequencies agreed with expected values. Also demonstrated was the unique capability to selectively enhance or suppress modes independently. For example, the ratio of the vibrational amplitudes of the 175 Hz first-bending and 1.33 kHz torsional modes of a small cantilever could be changed from in excess of 10:1 to less than 1:10 by shifting the ultrasound modulation phase 90 degrees. Similar changes were obtained for a 3 mm square MEMS mirror in the ratios of vibration amplitude around its two separate axes. Torsional modes of a hard-drive suspension could be selectively enhanced by over a factor of two by moving the ultrasound focus point from near the center to near the edge of the suspension.

In this paper, we present recent developments in the characterization of anisotropic, biological media utilizing displacements measured with dynamic MRE. In one approach, a waveguide constrained analysis is applied to physical structures (such as muscle), which evaluates the velocities of wave propagation along arbitrarily oriented fibers or fiber bundles in a local fashion, utilizing a spectral filter and a sliding window, spatial Fourier transform. In a second approach, the anisotropic equations of elasticity, in variational form, are inverted for a determination of the complex moduli comprising the medium. Issues concerning ill-conditioning, the effects of boundary conditions, and multiaspect excitation requirements will be addressed, as well as a discussion concerning the models which are appropriate for various physical situations. These two complementary methods of analysis will be demonstrated utilizing both phantom as well as in vivo dynamic displacement measurements.

The five string banjo is a stringed instrument that uses a drum head (membrane) to radiate sound. The strings couple to the head through a moveable bridge. Many have a removable resonator whose spacing from the main part of the banjo is adjustable to allow tuning of the internal air cavity. Holographic interferometry demonstrates vibrational modes of the head that have a strong dependence on head tension. Driving point accelerance measurements demonstrate that the ability of the bridge to be vibrated depends on the mass and stiffness of the materials used in its construction. Peak accelerances usually occur between 1800–2000 Hz. Power spectra measurements of the sound show that 99% of the sound is in a frequency range of 147–5200 Hz. Two substantial formants are seen in the power spectra. The first and largest occurs in about the 400–1800 Hz range, the same frequency range where the most substantial head modes are found. The second is from 2000–4000 Hz, a range where the series combination of bridge and head show increasing accelerances by driving point measurements. Measurements of the transient response following a pluck quantify the frequency content of the rising and falling phase.

The goal of our research is to develop MRI-based methods for assessing the mechanical properties of tissues in vivo. We have focused on a novel MRI technique for visualizing propagating acoustic shear waves [Science 269, 1854–1857 (1995)]. Suitable dynamic shear stress for Magnetic Resonance Elastography (MRE) can be generated by surface drivers, inertial effects, acoustic radiation pressure, or endogenous physiologic mechanisms. The MRE acquisition sequence is capable of visualizing cyclic tissue motion of less than 1 micron in displacement amplitude, with imaging times ranging from 100 ms to several minutes. Inversion algorithms based on continuum mechanics are used to process the acquired data to generate maps of mechanical properties such as depict stiffness, viscosity, attenuation, and anisotropic behavior. We have applied MRE to assess specimens of a variety of tissues, ranging in stiffness from lung to cartilage. Human studies have demonstrated that it is feasible to apply MRE to quantitatively image the mechanical properties of skeletal muscles, gray and white matter in the brain, thyroid, kidney, liver, and skin. Our preliminary clinical studies have to date applied MRE to observe changes in tissue mechanical properties in patients with breast, brain, and thyroid tumors, liver fibrosis, and diffuse diseases of skeletal muscle.