High Acoustic Impedance and Attenuation Backing for High-Frequency Focused P(VDF-TrFE)-Based Transducers.

Backing materials with tailored acoustic properties are beneficial for miniaturized ultrasonic transducer design. Whereas piezoelectric P(VDF-TrFE) films are common elements in high-frequency (>20 MHz) transducer design, their low coupling coefficient limits their sensitivity. Defining a suitable sensitivity-bandwidth trade-off for miniaturized high-frequency applications requires backings with impedances of >25 MRayl and strongly attenuating to account for miniaturized requirements. The motivation of this work is related to several medical applications such as small animal, skin or eye imaging. Simulations showed that increasing the acoustic impedance of the backing from 4.5 to 25 MRayl increases transducer sensitivity by 5 dB but decreases the bandwidth, which nevertheless remains high enough for the targeted applications. In this paper, porous sintered bronze material with spherically shaped grains, size-adapted for 25-30 MHz frequency, was impregnated with tin or epoxy resin to create multiphasic metallic backings. Microstructural characterizations of these new multiphasic composites showed that impregnation was incomplete and that a third air phase was present. The selected composites, sintered bronze-tin-air and sintered bronze-epoxy-air, at 5-35 MHz characterization, produced attenuation coefficients of 1.2 and >4 dB/mm/MHz and impedances of 32.4 and 26.4 MRayl, respectively. High-impedance composites were adopted as backing (thickness = 2 mm) to fabricate focused single-element P(VDF-TrFE)-based transducers (focal distance = 14 mm). The center frequency was 27 MHz, while the bandwidth at -6 dB was 65% for the sintered-bronze-tin-air-based transducer. We evaluated imaging performance using a pulse-echo system on a tungsten wire (diameter = 25 µm) phantom. Images confirmed the viability of integrating these backings in miniaturized transducers for imaging applications.

Topical delivery of mitogen-activated protein kinase inhibitor binimetinib prevents the development of cutaneous neurofibromas in neurofibromatosis type 1 mutant mice.

Cutaneous neurofibromas (cNFs) are a hallmark of patients with the neurofibromatosis type 1 (NF1) genetic disorder. These benign nerve sheath tumors, which can amount to thousands, develop from puberty onward, often cause pain and are considered by patients to be the primary burden of the disease. Mutations of NF1, encoding a negative regulator of the RAS signaling pathway, in the Schwann cell (SCs) lineage are considered to be at the origin of cNFs. The mechanisms governing cNFs development are poorly understood, and therapeutics to reduce cNFs are missing, mainly due to the lack of appropriate animal models. To address this, we designed the Nf1-KO mouse model that develops cNFs. Using this model, we found that cNFs development is a singular event and goes through 3 successive stages: initiation, progression, and stabilization characterized by changes in the proliferative and MAPK activities of tumor SCs. We found that skin trauma accelerated the development of cNFs and further used this model to explore the efficacy of the MEK inhibitor binimetinib to cure these tumors. We showed that while topically delivered binimetinib has a selective and minor effect on mature cNFs, the same drug prevents their development over long periods.

Inkjet-printed P(VDF-TrFE) film for high-frequency annular array.

An innovative processing to deposit P(VDF-TrFE) film on silicon wafers by an inkjet printing method was used to fabricate high-frequency annular array prototype. This prototype has a total aperture of 7.3 mm and 8 active elements. A polymer-based lens with low acoustic attenuation was added to the flat deposition on the wafer, setting the geometric focus to 13.8 mm. With a thickness of around 11 µm, the electromechanical performance of P(VDF-TrFE) films was evaluated with an effective thickness coupling factor of 22%. Electronics allowing all elements to simultaneously emit as a single element transducer was developed. In reception, a dynamic focusing, based on eight independent amplifying channels, was preferred. The center frequency of the prototype was 21.3 MHz, the insertion loss was 48.5 dB and the -6 dB fractional bandwidth was 143%. The trade-off sensitivity/bandwidth has rather favored the large bandwidth. Dynamic focusing on reception was applied and allowed to improvements in the lateral-full width at half maximum as shown on images obtained with a wire phantom at several depths. The next step, for a fully operational multi-element transducer, will be to achieve a significant increase of the acoustic attenuation in the silicon wafer.

Acoustic and Elastic Properties of a Blood Clot during Microbubble-Enhanced Sonothrombolysis: Hardening of the Clot with Inertial Cavitation.

Stroke is the second leading cause of death worldwide. Existing therapies present limitations, and other therapeutic alternatives are sought, such as sonothrombolysis with microbubbles (STL). The aim of this study was to evaluate the change induced by STL with or without recombinant tissue-type plasminogen activator (rtPA) on the acoustic and elastic properties of the blood clot by measuring its sound speed (SoS) and shear wave speed (SWS) with high frequency ultrasound and ultrafast imaging, respectively. An in-vitro setup was used and human blood clots were submitted to a combination of microbubbles and rtPA. The results demonstrate that STL induces a raise of SoS in the blood clot, specifically when combined with rtPA (p < 0.05). Moreover, the combination of rtPA and STL induces a hardening of the clot in comparison to rtPA alone (p < 0.05). This is the first assessment of acoustoelastic properties of blood clots during STL. The combination of rtPA and STL induce SoS and hardening of the clot, which is known to impair the penetration of thrombolytic drugs and their efficacy.

Cutaneous neurofibromas: patients' medical burden, current management and therapeutic expectations: results from an online European patient community survey.

BACKGROUND: Neurofibromatosis type 1 is an inherited condition with variable phenotypic expression and a high medical and social burden. The objectives of this patient survey were to better understand the real-world experiences of patients living with cutaneous neurofibromas (cNF), to perceive their satisfaction and feelings about cNF current management (only laser and surgery are currently available), and to highlight their expectations of new therapeutic modalities. RESULTS: One hundred seventy patients from 4 European countries took part in the study, 65% (n = 110) were women and mean age was 39 years old. 96% (n = 164) of respondents have cNF on visible parts of the body and the survey confirmed that total number of cNF and visibility increase with age. Patients reported that cNF mainly impacts everyday mood, general daily life and social life. The visibility of cNF had a higher impact than their number. 92% (n = 156) of patients have a regular and multidisciplinary medical follow-up. The dermatologist is one of the most consulted healthcare professionals. 76% (n = 130) of respondents have treated their cNF: 65% (n = 111) had surgery and 38% (n = 64) had multiple laser sessions. Frequency of operations and regrowth of cNF were the two most unsatisfactory aspects with both treatments for patients. Indeed, after removal, new cNF appear in more than 75% (n = 128) of cases. As a future treatment, patients expected a topical (30%, n = 51) or oral medication (29%, n = 50). Around 2 out of 3 patients would agree to take it at least once a day or more for life but they would like a well-tolerated treatment. According to patients, the most important effectiveness criteria of a new treatment are to block cNF growth and reduce their number. 70% (n = 119) of patients would consider a future treatment moderately effective to very effective if it could clear 30% of cNF. CONCLUSIONS: This first cNF European patient community survey confirmed that the visible stigma and unaesthetic aspect of cNF have an important impact on patients' quality of life. The survey highlighted that patients were not entirely satisfied with the actual surgery and laser treatments and revealed their clear and realistic expectations for future treatment of cNF.

A Novel Microflow Phantom Dedicated to Ultrasound Microvascular Measurements.

Tumor microvascularization is a biomarker of response to antiangiogenic treatments and is accurately assessed by ultrasound imaging. Imaging modes used to visualize slow flows include Power Doppler imaging, dynamic contrast-enhanced ultrasonography, and more recently, microvascular Doppler. Flow phantoms are used to evaluate the performance of Doppler imaging techniques, but they do not have a steady flow and sufficiently small channels. We report a novel device for robust and stable microflow measurements and the study of the microvascularization. Based on microfluidics technology, the prototype features wall-less cylindrical channels of diameters ranging from as small as 147 up to 436 µm, cast in a soft silicone polymer and perfused via a microfluidic flow pressure controller. The device was assessed using flow rates from 49 to 146 µL/min, with less than 1% coefficient of variation over three minutes, corresponding to velocities of 6 to 142 mm/s. This enabled us to evaluate and confirm the reliability of the Superb Microvascular Imaging Doppler mode compared with the Power Doppler mode at these flow rates in the presence of vibrations mimicking physiological motion.

3-D-Printed Phantom Fabricated by Photopolymer Jetting Technology for High-Frequency Ultrasound Imaging.

In the field of high-frequency ultrasound imaging ( MHz), tools for characterizing the performance of imaging systems are lacking. Indeed, commercial phantoms are often inadequate for this frequency range. The development of homemade phantoms on the laboratory scale is often required but is hindered by the difficulty in making very small structures that must be distributed with high accuracy in 3-D space. We propose investigating the use of 3-D photopolymer printing to create resolution and calibration phantoms designed for high-frequency ultrasound imaging. The quality and importance of these phantoms are discussed from the point of view of ultrasound parameters and imaging. First, the compressional wave group velocity, acoustic impedance, and attenuation of six photopolymerized materials were measured using temporal and spectral methods in a substitution experimental setup. Measurements were performed on printed samples using a broadband-focused single-element transducer covering a large frequency range (15-55 MHz). Two 3-D phantoms incorporating different shapes and dimensions were designed and printed. Finally, 3-D acoustic images were obtained using either a mechanically driven single-element transducer or a high-frequency commercial imaging system. Three-dimensional printing enabled us to generate phantoms suitable for high-frequency imaging with complex geometry inclusions and with a surrounding material having acoustic properties close to those of human skin. The calculated SNR between the inclusion and surrounding media is approximately 50 dB. In conclusion, 3-D printing is a useful tool for directly, easily, and rapidly manufacturing ultrasound phantoms for ultrasound imaging system assessments and computational calibration or validation.

Evaluation of high resolution ultrasound as a tool for assessing the 3D volume of blood clots during in vitro thrombolysis.

Thrombosis is a major cause of several diseases, i.e. myocardial infarction, cerebral stroke and pulmonary embolism. Thrombolytic therapies are required to induce fast and efficient recanalization of occluded vessels. To evaluate the in vitro efficacy of these thrombolytic strategies, measuring clot dissolution is essential. This study aimed to evaluate and validate high resolution ultrasound as a tool to assess the exact volume of clots in 3D and in real time during in vitro thrombolytic drug testing. This new method was validated by measuring the effects of concentration range of recombinant tissue type plasminogen activator on a blood clot during complete occlusion or 70% stenosis of a vessel. This study shows that high resolution ultrasound imaging allows for a real-time assessment of the 3D volume of a blood clot with negligible inter- and intra-operator variabilities. The conclusions drawn from this study demonstrate the promising potential of high resolution ultrasound imaging for the in vitro assessment of new thrombolytic drugs.

Anatomy and relations of the infraspinatus and the teres minor muscles: a fresh cadaver dissection study.

PURPOSE: Despite their functional importance, the infraspinatus (ISP) and teres minor (TM) muscles have been little investigated. This study aimed to describe the macroscopic morphology, innervation, and inter-relations of the ISP and TM muscles. METHODS: Forty fresh cadaver dissections and histologic analysis were performed. Three groups of specimens were distinguished according to the rotator cuff tendon status: (1) intact rotator cuff; (2) supraspinatus tendon tears with intact ISP tendon; and (3) both supraspinatus and ISP tendons torn. Muscle fiber organization and muscle and tendon length were recorded. ISP and TM innervation and fiber structure were studied. RESULTS: ISP muscles were composed of three groups of fiber organized in two planes: two superficial groups, with mean pennation angles of, respectively, 27° ± 4° and 23° ± 3° with respect to the axis of the central tendon of the underlying group. TMs were thick fusiform muscles showing a parallel organization; 26 specimens (67 %) had aponeuroses isolating the TM, with a mean length of 5.2 ± 2.7 cm. Rotator cuff lesions were associated with relatively greater ISP tendon than muscle length. Innervation of the ISP muscle comprised 2-4 main branches from the suprascapular nerve and that of the TM 1 branch from the axillary nerve. CONCLUSION: ISP muscle body morphology derives from three groups of fibers in two planes. The TM has a parallel organization. Several nerve branches innervate the ISP muscle, whereas only one supplies the TM. The limits between the two muscles bodies consist of an aponeurotic fascia in two-thirds of cases.

Identification of epigenetic factors regulating the mesenchyme to epithelium transition by RNA interference screening in breast cancer cells.

BACKGROUND: In breast cancer, the epithelial to mesenchyme transition (EMT) is associated to tumour dissemination, drug resistance and high relapse risks. It is partly controlled by epigenetic modifications such as histone acetylation and methylation. The identification of genes involved in these reversible modifications represents an interesting therapeutic strategy to fight metastatic disease by inducing mesenchymal cell differentiation to an epithelial phenotype. METHODS: We designed a siRNA library based on chromatin modification-related to functional domains and screened it in the mesenchymal breast cancer cell line MDA-MB-231. The mesenchyme to epithelium transition (MET) activation was studied by following human E-CADHERIN (E-CAD) induction, a specific MET marker, and cell morphology. Candidate genes were validated by studying the expression of several differential marker genes and their impact on cell migration. RESULTS: The screen led to the identification of 70 gene candidates among which some are described to be, directly or indirectly, involved in EMT like ZEB1, G9a, SMAD5 and SMARCD3. We also identified the DOT1L as involved in EMT regulation in MDA-MB-231. Moreover, for the first time, KAT5 gene was linked to the maintenance of the mesenchymal phenotype. CONCLUSIONS: A multi-parametric RNAi screening approach was developed to identify new EMT regulators such as KAT5 in the triple negative breast cancer cell line MDA-MB-231.

Design and synthesis of new non nucleoside inhibitors of DNMT3A.

DNA methylation, an epigenetic modification regulating gene expression, is a promising target in cancer. In an effort to identify new non nucleosidic inhibitors of DNA methyltransferases, the enzymes responsible for DNA methylation, we carried out a high-throughput screening of 66,000 chemical compounds based on an enzymatic assay against catalytic DNMT3A. A family of propiophenone derivatives was identified. After chemical optimization and structure activity relationship studies, a new inhibitor (33) was obtained with an EC50 of 2.1 µM against DNMT3A. The mechanism of inhibition of the compound was investigated as it forms a reactive Michael acceptor group in situ. Thereby, the Michael acceptor 20 was identified. This compound was further characterized for its biological activity in cancer cells.

Evaluation of bias voltage modulation sequence for nonlinear contrast agent imaging using a capacitive micromachined ultrasonic transducer array.

Many clinical diagnoses have now been improved thanks to the development of new techniques dedicated to contrast agent nonlinear imaging. Over the past few years, Capacitive Micromachined Ultrasonic Transducers (cMUTs) have emerged as a promising alternative to traditional piezoelectric transducers. One notable advantage of cMUTs is their wide frequency bandwidth. However, their use in nonlinear imaging approaches such as those used to detect contrast agents have been challenging due their intrinsic nonlinear character. We propose a new contrast imaging sequence, called bias voltage modulation (BVM), specifically developed for cMUTs to suppress their inherent nonlinear behavior. Theoretical and experimental results show that a complete cancellation of the nonlinear signal from the source can be reached when the BVM sequence is implemented. In-vitro validation of the sequence is performed using a cMUT probe connected to an open scanner and a flow phantom setup containing SonoVue microbubbles. Compared to the standard amplitude modulation imaging mode, a 6 dB increase of contrast-to-tissue ratio was achieved when the BVM sequence is applied. These results reveal that the problem of cMUT nonlinearity can be addressed, thus expanding the potential of this new transducer technology for nonlinear contrast agent detection and imaging.

Design, synthesis and biological evaluation of 4-amino-N- (4-aminophenyl)benzamide analogues of quinoline-based SGI-1027 as inhibitors of DNA methylation.

Quinoline derivative SGI-1027 (N-(4-(2-amino-6-methylpyrimidin-4-ylamino)phenyl)-4-(quinolin-4-ylamino)benzamide) was first described in 2009 as a potent inhibitor of DNA methyltransferase (DNMT) 1, 3A and 3B. Based on molecular modeling studies, performed using the crystal structure of Haemophilus haemolyticus cytosine-5 DNA methyltransferase (MHhaI C5 DNMT), which suggested that the quinoline and the aminopyridimine moieties of SGI-1027 are important for interaction with the substrates and protein, we designed and synthesized 25 derivatives. Among them, four compounds­namely the derivatives 12, 16, 31 and 32­exhibited activities comparable to that of the parent compound. Further evaluation revealed that these compounds were more potent against human DNMT3A than against human DNMT1 and induced the re-expression of a reporter gene, controlled by a methylated cytomegalovirus (CMV) promoter, in leukemia KG-1 cells. These compounds possessed cytotoxicity against leukemia KG-1 cells in the micromolar range, comparable with the cytotoxicity of the reference compound, SGI-1027. Structure­activity relationships were elucidated from the results. First, the presence of a methylene or carbonyl group to conjugate the quinoline moiety decreased the activity. Second, the size and nature of the aromatic or heterocycle subsitutents effects inhibition activity: tricyclic moieties, such as acridine, were found to decrease activity, while bicyclic substituents, such as quinoline, were well tolerated. The best combination was found to be a bicyclic substituent on one side of the compound, and a one-ring moiety on the other side. Finally, the orientation of the central amide bond was found to have little effect on the biological activity. This study provides new insights in to the structure-activity relationships of SGI-1027 and its derivative.

Lead zirconate titanate-based thick films for high-frequency focused ultrasound transducers prepared by electrophoretic deposition.

An electrophoretic deposition (EPD) process with high deposition rate was used to fabricate a curved piezoelectric thick film devoted to high-frequency transducers for medical imaging. Niobium-doped lead zirconate titanate (PZTNb) powder was stabilized in ethanol to prepare a suspension with high zeta potential and low conductivity. A gold layer, pad-printed and fired on a curved porous PZT substrate, was used as the working electrode for the deposition of the PZTNb thick film. This substrate was chosen because it has the required properties (acoustic impedance and attenuation) to be used directly as a backing for the high-frequency transducer, leading to a simplified process for transducer assembly with this integrated structure. PZT-Nb thick films were also deposited by EPD on flat gold-coated alumina substrates as a reference. The thickness of the films was between 20 and 35 µm, and their electromechanical performance was comparable to standard PZT bulk ceramics with a thickness coupling factor of 48%. For the curved thick film, the thickness coupling factor was slightly lower. The corresponding integrated structure was used to fabricate a transducer with a center frequency of 40 MHz and an f-number of 2.8. It was integrated into a realtime ultrasound scanner and used to image human forearm skin; the resulting images showed, for the first time, the efficacy of the EPD process for these imaging applications.

Dual-frequency transducer for nonlinear contrast agent imaging.

Detection of high-order nonlinear components issued from microbubbles has emerged as a sensitive method for contrast agent imaging. Nevertheless, the detection of these high-frequency components, including the third, fourth, and fifth harmonics, remains challenging because of the lack of transducer sensitivity and bandwidth. In this context, we propose a new design of imaging transducer based on a simple fabrication process for high-frequency nonlinear imaging. The transducer is composed of two elements: the outer low-frequency (LF) element was centered at 4 MHz and used in transmit mode, whereas the inner high-frequency (HF) element centered at 14 MHz was used in receive mode. The center element was pad-printed using a lead zirconate titanate (PZT) paste. The outer element was molded using a commercial PZT, and curved porous unpoled PZT was used as backing. Each piezoelectric element was characterized to determine the electromechanical performance with thickness coupling factor around 45%. After the assembly of the two transducer elements, hydrophone measurements (electroacoustic responses and radiation patterns) were carried out and demonstrated a large bandwidth (70% at -3 dB) of the HF transducer. Finally, the transducer was evaluated for contrast agent imaging using contrast agent microbubbles. The results showed that harmonic components (up to the sixth harmonic) of the microbubbles were successfully detected. Moreover, images from a flow phantom were acquired and demonstrated the potential of the transducer for high-frequency nonlinear contrast imaging.

DNA methylation inhibitors in cancer: recent and future approaches.

This review presents the different human DNA methyltransferases (DNMTs), their biological roles, their mechanisms of action and their role in cancer. The description of assays for detecting DNMT inhibitors (DNMTi) follows. The different known DNMTi are reported along with their advantages, drawbacks and clinical trials. A discussion on the features of the future DNMT inhibitors will conclude this review.