Genome-wide cancer-specific chromatin accessibility patterns derived from archival processed xenograft tumors.

Chromatin accessibility states that influence gene expression and other nuclear processes can be altered in disease. The constellation of transcription factors and chromatin regulatory complexes in cells results in characteristic patterns of chromatin accessibility. The study of these patterns in tissues has been limited because existing chromatin accessibility assays are ineffective for archival formalin-fixed, paraffin-embedded (FFPE) tissues. We have developed a method to efficiently extract intact chromatin from archival tissue via enhanced cavitation with a nanodroplet reagent consisting of a lipid shell with a liquid perfluorocarbon core. Inclusion of nanodroplets during the extraction of chromatin from FFPE tissues enhances the recovery of intact accessible and nucleosome-bound chromatin. We show that the addition of nanodroplets to the chromatin accessibility assay formaldehyde-assisted isolation of regulatory elements (FAIRE), does not affect the accessible chromatin signal. Applying the technique to FFPE human tumor xenografts, we identified tumor-relevant regions of accessible chromatin shared with those identified in primary tumors. Further, we deconvoluted non-tumor signal to identify cellular components of the tumor microenvironment. Incorporation of this method of enhanced cavitation into FAIRE offers the potential for extending chromatin accessibility to clinical diagnosis and personalized medicine, while also enabling the exploration of gene regulatory mechanisms in archival samples.

Element Position Calibration for Matrix Array Transducers with Multiple Disjoint Piezoelectric Panels.

Two-dimensional ultrasound transducers enable the acquisition of fully volumetric data that have been demonstrated to provide greater diagnostic information in the clinical setting and are a critical tool for emerging ultrasound methods, such as super-resolution and functional imaging. This technology, however, is not without its limitations. Due to increased fabrication complexity, some matrix probes with disjoint piezoelectric panels may require initial calibration. In this manuscript, two methods for calibrating the element positions of the Vermon 1024-channel 8 MHz matrix transducer are detailed. This calibration is a necessary step for acquiring high resolution B-mode images while minimizing transducer-based image degradation. This calibration is also necessary for eliminating vessel-doubling artifacts in super-resolution images and increasing the overall signal-to-noise ratio (SNR) of the image. Here, we show that the shape of the point spread function (PSF) can be significantly improved and PSF-doubling artifacts can be reduced by up to 10 dB via this simple calibration procedure.

A fully automated algorithm for heart rate detection in post-dive precordial Doppler ultrasound.

Background: Doppler ultrasound is used currently in decompression research for the evaluation of venous gas emboli (VGE). Estimation of heart rate from post-dive Doppler ultrasound recordings can provide a tool for the evaluation of physiological changes from decompression stress, as well as aid in the development of automated VGE detection algorithms that relate VGE presence to cardiac activity. Method: An algorithm based on short-term autocorrelation was developed in MATLAB to estimate the heart rate in post-dive precordial Doppler ultrasound. The algorithm was evaluated on 21 previously acquired and labeled precordial recordings spanning Kisman-Masurel (KM) codes of 111-444 (KM I-IV) with manually derived instantaneous heart rates. Results: A window size of at least two seconds was necessary for robust and accurate instantaneous heart rate estimation with a mean error of 1.56 ± 7.10 bpm. Larger window sizes improved the algorithm performance, at the cost of beat-to-beat heart rate estimates. We also found that our algorithm provides good results for low KM grade Doppler recordings with and without flexion, and high KM grades without flexion. High KM grades observed after movement produced the greatest mean absolute error of 6.12 ± 8.40 bpm. Conclusion: We have developed a fully automated algorithm for the estimation of heart rate in post-dive precordial Doppler ultrasound recordings.

Low-boiling Point Perfluorocarbon Nanodroplets as Dual-Phase Dual-Modality MR/US Contrast Agent.

Detection of bare gas microbubbles by magnetic resonance (MR) at low concentrations typically used in clinical contrast-ultrasound studies was recently demonstrated using hyperCEST. Despite the enhanced sensitivity achieved with hyperCEST, in vivo translation is challenging as on-resonance saturation of the gas-phase core of microbubbles consequently results in saturation of the gas-phase hyperpolarized 129 Xe within the lungs. Alternatively, microbubbles can be condensed into the liquid phase to form perfluorocarbon nanodroplets, where 129 Xe resonates at a chemical shift that is separated from the gas-phase signal in the lungs. For ultrasound applications, nanodroplets can be acoustically reverted back into their microbubble form to act as a phase-change contrast agent. Here, we show that low-boiling point perfluorocarbons, both in their liquid and gas form, generate phase-dependent hyperCEST contrast. Magnetic resonance detection of ultrasound-mediated phase transition demonstrates that these perfluorocarbons could be used as a dual-phase dual-modality MR/US contrast agent.

Focused Ultrasound for Immunomodulation of the Tumor Microenvironment.

Focused ultrasound (FUS) has recently emerged as a modulator of the tumor microenvironment, paving the way for FUS to become a safe yet formidable cancer treatment option. Several mechanisms have been proposed for the role of FUS in facilitating immune responses and overcoming drug delivery barriers. However, with the wide variety of FUS parameters used in diverse tumor types, it is challenging to pinpoint FUS specifications that may elicit the desired antitumor response. To clarify FUS bioeffects, we summarize four mechanisms of action, including thermal ablation, hyperthermia/thermal stress, mechanical perturbation, and histotripsy, each inducing unique vascular and immunological effects. Notable tumor responses to FUS include enhanced vascular permeability, increased T cell infiltration, and tumor growth suppression. In this review, we have categorized and reviewed recent methods of using therapeutic ultrasound to elicit an antitumor immune response with examples that reveal specific solutions and challenges in this new research area.

A Rapid Prototyping Method for Sub-MHz Single-Element Piezoelectric Transducers by Using 3D-Printed Components.

We present a rapid prototyping method for sub-megahertz single-element piezoelectric transducers by using 3D-printed components. In most of the early research phases of applying new sonication ideas, the prototyping quickness is prioritized over the final packaging quality, since the quickness of preliminary demonstration is crucial for promptly determining specific aims and feasible research approaches. We aim to develop a rapid prototyping method for functional ultrasonic transducers to overcome the current long lead time (>a few weeks). Here, we used 3D-printed external housing parts considering a single matching layer and either air backing or epoxy-composite backing (acoustic impedance > 5 MRayl). By molding a single matching layer on the top surface of a piezoceramic in a 3D-printed housing, an entire packaging time was significantly reduced (<26 h) compared to the conventional methods with grinding, stacking, and bonding. We demonstrated this prototyping method for 590-kHz single-element, rectangular-aperture transducers for moderate pressure amplitudes (mechanical index > 1) at focus with temporal pulse controllability (maximum amplitude by <5-cycle burst). We adopted an air-backing design (Type A) for efficient pressure outputs, and bandwidth improvement was tested by a tungsten-composite-backing (Type B) design. The acoustic characterization results showed that the type A prototype provided 3.3 kPa/Vpp far-field transmitting sensitivity with 25.3% fractional bandwidth whereas the type B transducer showed 2.1 kPa/Vpp transmitting sensitivity with 43.3% fractional bandwidth. As this method provided discernable quickness and cost efficiency, this detailed rapid prototyping guideline can be useful for early-phase sonication projects, such as multi-element therapeutic ultrasound array and micro/nanomedicine testing benchtop device prototyping.

Magnetic Resonance Detection of Gas Microbubbles via HyperCEST: A Path Toward Dual Modality Contrast Agent.

Gas microbubbles are an established clinical ultrasound contrast agent. They could also become a powerful magnetic resonance (MR) intravascular contrast agent, but their low susceptibility-induced contrast requires high circulating concentrations or the addition of exogenous paramagnetic nanoparticles for MR detection. In order to detect clinical in vivo concentrations of raw microbubbles via MR, an alternative detection scheme must be used. HyperCEST is an NMR technique capable of indirectly detecting signals from very dilute molecules (concentrations well below the NMR detection threshold) that exchange hyperpolarized 129 Xe. Here, we use quantitative hyperCEST to show that microbubbles are very efficient hyperCEST agents. They can accommodate and saturate millions of 129 Xe atoms at a time, allowing for their indirect detection at concentrations as low as 10 femtomolar. The increased MR sensitivity to microbubbles achieved via hyperCEST can bridge the gap for microbubbles to become a dual modality contrast agent.

Ultrasound in decompression research: fundamentals, considerations, and future technologies.

It is widely accepted that bubbles are a necessary but insufficient condition for the development of decompression sickness. However, open questions remain regarding the precise formation and behavior of these bubbles after an ambient pressure reduction (decompression), primarily due to the inherent difficulty of directly observing this phenomenon in vivo. In decompression research, information about these bubbles after a decompression is gathered via means of ultrasound acquisitions. The ability to draw conclusions regarding decompression research using ultrasound is highly influenced by the variability of the methodologies and equipment utilized by different research groups. These differences play a significant role in the quality of the data and thus the interpretation of the results. The purpose of this review is to provide a technical overview of the use of ultrasound in decompression research, particularly Doppler and brightness (B)-mode ultrasound. Further, we will discuss the strengths and limitations of these technologies and how new advancements are improving our ability to understand bubble behavior post-decompression.

A fully automated method for late ventricular diastole frame selection in post-dive echocardiography without ECG gating.

Venous gas emboli (VGE) are often quantified as a marker of decompression stress on echocardiograms. Bubble-counting has been proposed as an easy to learn method, but remains time-consuming, rendering large dataset analysis impractical. Computer automation of VGE counting following this method has therefore been suggested as a means to eliminate rater bias and save time. A necessary step for this automation relies on the selection of a frame during late ventricular diastole (LVD) for each cardiac cycle of the recording. Since electrocardiograms (ECG) are not always recorded in field experiments, here we propose a fully automated method for LVD frame selection based on regional intensity minimization. The algorithm is tested on 20 previously acquired echocardiography recordings (from the original bubble-counting publication), half of which were acquired at rest (Rest) and the other half after leg flexions (Flex). From the 7,140 frames analyzed, sensitivity was found to be 0.913 [95% CI: 0.875-0.940] and specificity 0.997 [95% CI: 0.996-0.998]. The method's performance is also compared to that of random chance selection and found to perform significantly better (p≺0.0001). No trend in algorithm performance was found with respect to VGE counts, and no significant difference was found between Flex and Rest (p>0.05). In conclusion, full automation of LVD frame selection for the purpose of bubble counting in post-dive echocardiography has been established with excellent accuracy, although we caution that high quality acquisitions remain paramount in retaining high reliability.

On Command Drug Delivery via Cell-Conveyed Phototherapeutics.

Herein, the use of red blood cells (RBCs) as carriers of cytoplasmically interned phototherapeutic agents is described. Photolysis promotes drug release from the RBC carrier thereby providing the means to target specific diseased sites. This strategy is realized with a vitamin B12-taxane conjugate (B12-TAX), in which the drug is linked to the vitamin via a photolabile CoC bond. The conjugate is introduced into mouse RBCs (mRBCs) via a pore-forming/pore-resealing procedure and is cytoplasmically retained due to the membrane impermeability of B12. Photolysis separates the taxane from the B12 cytoplasmic anchor, enabling the drug to exit the RBC carrier. A covalently appended Cy5 antenna sensitizes the conjugate (Cy5-B12-TAX) to far red light, thereby circumventing the intense light absorbing properties of hemoglobin (350-600 nm). Microscopy and imaging flow cytometry reveal that Cy5-B12-TAX-loaded mRBCs act as drug carriers. Furthermore, intravital imaging of mice furnish a real time assessment of circulating phototherapeutic-loaded mRBCs as well as evidence of the targeted photorelease of the taxane upon photolysis. Histopathology confirms that drug release occurs in a well resolved spatiotemporal fashion. Finally, acoustic angiography is employed to assess the consequences of taxane release at the tumor site in Nu/Nu-tumor-bearing mice.

Cavitation Enhancement Increases the Efficiency and Consistency of Chromatin Fragmentation from Fixed Cells for Downstream Quantitative Applications.

One of the most sensitive, time-consuming, and variable steps of chromatin immunoprecipitation (ChIP) is chromatin sonication. Traditionally, this process can take hours to properly sonicate enough chromatin for multiple ChIP assays. Further, the length of sheared DNA is often inconsistent. In order to faithfully measure chemical and structural changes at the chromatin level, sonication needs to be reliable. Thus, chromatin fragmentation by sonication represents a significant bottleneck to downstream quantitative analysis. To improve the consistency and efficiency of chromatin sonication, we developed and tested a cavitation enhancing reagent based on sonically active nanodroplets. Here, we show that nanodroplets increase sonication efficiency by 16-fold and provide more consistent levels of chromatin fragmentation. Using the previously characterized chromatin in vivo assay (CiA) platform, we generated two distinct chromatin states in order to test nanodroplet-assisted sonication sensitivity in measuring post-translational chromatin marks. By comparing euchromatin to chemically induced heterochromatin at the same CiA:Oct4 locus, we quantitatively measure the capability of our new sonication technique to resolve differences in chromatin structure. We confirm that nanodroplet-assisted sonication results are indistinguishable from those of samples processed with traditional sonication in downstream applications. While the processing time for each sample was reduced from 38.4 to 2.3 min, DNA fragment distribution sizes were significantly more consistent with a coefficient of variation 2.7 times lower for samples sonicated in the presence of nanodroplets. In conclusion, sonication utilizing the nanodroplet cavitation enhancement reagent drastically reduces the amount of processing time and provides consistently fragmented chromatin of high quality for downstream applications.

A Pilot Clinical Study in Characterization of Malignant Renal-cell Carcinoma Subtype with Contrast-enhanced Ultrasound.

Malignant renal cell carcinoma (RCC) is a diverse set of diseases, which are independently difficult to characterize using conventional MRI and CT protocols due to low temporal resolution to study perfusion characteristics. Because different disease subtypes have different prognoses and involve varying treatment regimens, the ability to determine RCC subtype non-invasively is a clinical need. Contrast-enhanced ultrasound (CEUS) has been assessed as a tool to characterize kidney lesions based on qualitative and quantitative assessment of perfusion patterns, and we hypothesize that this technique might help differentiate disease subtypes. Twelve patients with RCC confirmed pathologically were imaged using contrast-enhanced ultrasound. Time intensity curves were generated and analyzed quantitatively using 10 characteristic metrics. Results showed that peak intensity ( p = 0.001) and time-to-80% on wash-out ( p = 0.004) provided significant differences between clear cell, papillary, and chromophobe RCC subtypes. These results suggest that CEUS may be a feasible test for characterizing RCC subtypes.

Wideband acoustic activation and detection of droplet vaporization events using a capacitive micromachined ultrasonic transducer.

An ongoing challenge exists in understanding and optimizing the acoustic droplet vaporization (ADV) process to enhance contrast agent effectiveness for biomedical applications. Acoustic signatures from vaporization events can be identified and differentiated from microbubble or tissue signals based on their frequency content. The present study exploited the wide bandwidth of a 128-element capacitive micromachined ultrasonic transducer (CMUT) array for activation (8 MHz) and real-time imaging (1 MHz) of ADV events from droplets circulating in a tube. Compared to a commercial piezoelectric probe, the CMUT array provides a substantial increase of the contrast-to-noise ratio.

FEASIBILITY AND SAFETY OF CONTRAST-ENHANCED ULTRASOUND IN THE DISTAL LIMB OF SIX HORSES.

Vascular alterations play important roles in many orthopedic diseases such as osteoarthritis, tendonitis, and synovitis in both human and equine athletes. Understanding these alterations could enhance diagnosis, prognosis, and treatment. Contrast-enhanced ultrasound (CEUS) could be a valuable method for evaluation of blood flow and perfusion of these processes in the equine distal limb, however no reports were found describing feasibility or safety of the technique. The goal of this prospective, experimental study was to describe the feasibility and safety of distal limb CEUS in a sample of six horses. For each horse, CEUS of the distal limb was performed after intravenous injections of 5 and 10 ml, as well as intra-arterial injections of 0.5 and 1 ml contrast medium. Vital parameters were monitored and CEUS images were assessed qualitatively and quantitatively for degree of contrast enhancement. None of the horses had clinically significant changes in their vital parameters after contrast medium injection. One horse had a transient increase in respiratory rate, and several horses had mild increases of systolic blood pressure of short duration after intravenous, but not after intra-arterial injections. Intra-arterial injection was possible in all horses and resulted in significantly improved contrast enhancement both quantitatively (P = 0.027) and qualitatively (P = 0.019). Findings from this study indicated that CEUS is a feasible and safe diagnostic test for evaluation of the equine distal limb. Future studies are needed to assess the clinical utility of this test for horses with musculoskeletal diseases.

Dual-frequency piezoelectric transducers for contrast enhanced ultrasound imaging.

For many years, ultrasound has provided clinicians with an affordable and effective imaging tool for applications ranging from cardiology to obstetrics. Development of microbubble contrast agents over the past several decades has enabled ultrasound to distinguish between blood flow and surrounding tissue. Current clinical practices using microbubble contrast agents rely heavily on user training to evaluate degree of localized perfusion. Advances in separating the signals produced from contrast agents versus surrounding tissue backscatter provide unique opportunities for specialized sensors designed to image microbubbles with higher signal to noise and resolution than previously possible. In this review article, we describe the background principles and recent developments of ultrasound transducer technology for receiving signals produced by contrast agents while rejecting signals arising from soft tissue. This approach relies on transmitting at a low-frequency and receiving microbubble harmonic signals at frequencies many times higher than the transmitted frequency. Design and fabrication of dual-frequency transducers and the extension of recent developments in transducer technology for dual-frequency harmonic imaging are discussed.

Nucleation and growth synthesis of siloxane gels to form functional, monodisperse, and acoustically programmable particles.

Nucleation and growth methods offer scalable means of synthesizing colloidal particles with precisely specified size for applications in chemical research, industry, and medicine. These methods have been used to prepare a class of silicone gel particles that display a range of programmable properties and narrow size distributions. The acoustic contrast factor of these particles in water is estimated and can be tuned such that the particles undergo acoustophoresis to either the pressure nodes or antinodes of acoustic standing waves. These particles can be synthesized to display surface functional groups that can be covalently modified for a range of bioanalytical and acoustophoretic sorting applications.

Design and Fabrication of 1-D CMUT Arrays for Dual-Mode Dual-Frequency Acoustic Angiography Applications.

When microbubble contrast agents are excited at low frequencies (less than 5 MHz), they resonate and produce higher-order harmonics due to their nonlinear behavior. We propose a novel scheme with a capacitive micromachined ultrasonic transducer (CMUT) array to receive high-frequency microbubble harmonics in collapse mode and to transmit a low-frequency high-pressure pulse by releasing the CMUT plate from collapse and pull it back to collapse again in the same transmit-receive cycle. By patterning and etching the substrate to create glass spacers in the device cavity we can reliably operate the CMUT in collapse mode and receive high-frequency signals. Previously, we demonstrated a single-element CMUT with spacers operating in the described fashion. In this article, we present the design and fabrication of a dual-mode, dual-frequency 1-D CMUT array with 256 elements. We present two different insulating glass spacer designs in rectangular cells for the collapse mode. For the device with torus-shaped spacers, the 3 dB receive bandwidth is from 8 to 17 MHz, and the transmitted maximum peak-to-peak pressure from 32 elements at 4 mm focal depth was 2.12 MPa with a 1.21 MPa peak negative pressure, which corresponds to a mechanical index (MI) of 0.58 at 4.3 MHz. For the device with line-shaped spacers, the 3-dB receive bandwidth at 150 V dc bias extends from 10.9 to 19.2 MHz. By increasing the bias voltage to 180 V, the 3 dB bandwidth shifts, and extends from 11.7 to 20.4 MHz. The transmitting maximum peak-to-peak pressure with 32 elements at 4 mm was 2.06 MPa with a peak negative pressure of 1.19 MPa, which corresponds to an MI of 0.62 at 3.7 MHz.

Current clinical investigations of focused ultrasound blood-brain barrier disruption: A review.

The blood-brain barrier (BBB) presents a formidable challenge in delivering therapeutic agents to the central nervous system. Ultrasound-mediated BBB disruption has emerged as a promising non-invasive technique to enhance drug delivery to the brain. This manuscript reviews fundamental principles of ultrasound-based techniques and their mechanisms of action in temporarily permeabilizing the BBB. Clinical trials employing ultrasound for BBB disruption are discussed, summarizing diverse applications ranging from the treatment of neurodegenerative diseases to targeted drug delivery for brain tumors. The review also addresses safety considerations, outlining the current understanding of potential risks and mitigation strategies associated with ultrasound exposure, including real-time monitoring and assessment of treatment efficacy. Among the large number of studies, significant successes are highlighted thus providing perspective on the future direction of the field.

In vivo demonstration of cancer molecular imaging with ultrasound radiation force and buried-ligand microbubbles.

In designing targeted contrast agent materials for imaging, the need to present a targeting ligand for recognition and binding by the target is counterbalanced by the need to minimize interactions with plasma components and to avoid recognition by the immune system. We have previously reported on a microbubble imaging probe for ultrasound molecular imaging that uses a buried-ligand surface architecture to minimize unwanted interactions and immunogenicity. Here we examine for the first time the utility of this approach for in vivo molecular imaging. In accordance with previous results, we showed a threefold increase in circulation persistence through the tumor of a fibrosarcoma model in comparison with controls. The buried-ligand microbubbles were then activated for targeted adhesion through the application of noninvasive ultrasound radiation forces applied specifically to the tumor region. Using a clinical ultrasound scanner, microbubbles were activated, imaged, and silenced. The results showed visually conspicuous images of tumor neovasculature and a twofold increase in ultrasound radiation force enhancement of acoustic contrast intensity for buried-ligand microbubbles, whereas no such increase was found for exposed-ligand microbubbles. We therefore conclude that the use of acoustically active buried-ligand microbubbles for ultrasound molecular imaging bridges the demand for low immunogenicity with the necessity of maintaining targeting efficacy and imaging conspicuity in vivo.

Microfluidic fabrication of stable gas-filled microcapsules for acoustic contrast enhancement.

We introduce a facile approach for the production of gas-filled microcapsules designed to withstand high pressures. We exploit microfluidics to fabricate water-filled microcapsules that are then externally triggered to become gas-filled, thus making them more echogenic. In addition, the gas-filled microcapsules have a solid polymer shell making them resistant to pressure-induced buckling, which makes them more mechanically robust than traditional prestabilized microbubbles; this should increase the potential of their utility for acoustic imaging of porous media with high hydrostatic pressures such as oil reservoirs.