Reconfigurable Split Ring Resonators by MEMS-Driven Geometrical Tuning.

A novel approach for dynamic microwave modulation is proposed in the form of reconfigurable resonant circuits. This result is obtained through the monolithic integration of double split ring resonators (DSRRs) with microelectromechanical actuators (MEMS) for geometrical tuning. Two configurations were analyzed to achieve a controlled deformation of the DSRRs' metamaterial geometry by mutual rotation or extrusion along the azimuthal direction of the two constituent rings. Then, the transfer function was numerically simulated for a reconfigurable MEMS-DSRR hybrid architecture where the DSRR is embedded onto a realistic piezo actuator chip. In this case, a 370 MHz resonance frequency shift was obtained under of a 170 µm extrusion driven by a DC voltage. These characteristics in combination with a high Q factor and dimensions compatible with standard CMOS manufacturing techniques provide a step forward for the production of devices with applications in multiband telecommunications and wireless power transfer and in the IoT field.

INSPEX: Optimize Range Sensors for Environment Perception as a Portable System.

Environment perception is crucial for the safe navigation of vehicles and robots to detect obstacles in their surroundings. It is also of paramount interest for navigation of human beings in reduced visibility conditions. Obstacle avoidance systems typically combine multiple sensing technologies (i.e., LiDAR, radar, ultrasound and visual) to detect various types of obstacles under different lighting and weather conditions, with the drawbacks of a given technology being offset by others. These systems require powerful computational capability to fuse the mass of data, which limits their use to high-end vehicles and robots. INSPEX delivers a low-power, small-size and lightweight environment perception system that is compatible with portable and/or wearable applications. This requires miniaturizing and optimizing existing range sensors of different technologies to meet the user's requirements in terms of obstacle detection capabilities. These sensors consist of a LiDAR, a time-of-flight sensor, an ultrasound and an ultra-wideband radar with measurement ranges respectively of 10 m, 4 m, 2 m and 10 m. Integration of a data fusion technique is also required to build a model of the user's surroundings and provide feedback about the localization of harmful obstacles. As primary demonstrator, the INSPEX device will be fixed on a white cane.

PMUTs Arrays for Structural Health Monitoring of Bolted-Joints.

Micro-electro-mechanical systems (MEMS) have enabled new techniques for the miniaturization of sensors suitable for Structural Health Monitoring (SHM) applications. In this study, MEMS-based sensors, specifically Piezoelectric Micromachined Ultrasonic Transducers (PMUT), are used to evaluate and monitor the pre-tensioning of a bolted joint structural system. For bolted joints to function properly, it is essential to maintain a suitable level of pre-tensioning. In this work, an array of PMUTs attached to the head and to the end of a bolt, serve as transmitter and receiver, respectively, in a pitch-catch Ultrasonic Testing (UT) scenario. The primary objective is to detect the Change in Time of Flight (CTOF) of the acoustic wave generated by the PMUT array and propagating along the bolt's axis between a non-loaded bolt and a bolt in service. To model the pre-tensioning of bolted joints and the transmission of the acoustic wave to and from a group of PMUTs through the bolt, a set of numerical models is created. The CTOF is found to be linearly related to the amount of pre-tensioning. The numerical model is validated through comparisons with the results of a preliminary experimental campaign.

Role of ribosome recycling factor in natural termination and translational coupling as a ribosome releasing factor.

The role of ribosome recycling factor (RRF) of E. coli was studied in vivo and in vitro. We used the translational coupling without the Shine-Dalgarno sequence of downstream ORF (d-ORF) as a model system of the RRF action in natural termination of protein synthesis. For the in vivo studies we used the translational coupling by the adjacent coat and lysis genes of RNA phage GA sharing the termination and initiation (UAAUG) and temperature sensitive RRF. The d-ORF translation was measured by the expression of the reporter lacZ gene connected to the 5'-terminal part of the lysis gene. The results showed that more ribosomes which finished upstream ORF (u-ORF) reading were used for downstream reading when RRF was inactivated. The in vitro translational coupling studies with 027mRNA having the junction sequence UAAUG with wild-type RRF were carried out with measuring amino acids incorporation. The results showed that ribosomes released by RRF read downstream from AUG of UAAUG. In the absence of RRF, ribosomes read downstream in frame with UAA. These in vivo and in vitro studies indicate that RRF releases ribosomes from mRNA at the termination codon of u-ORF. Furthermore, the non-dissociable ribosomes read downstream from AUG of UAAUG with RRF in vitro. This suggests that complete ribosomal splitting is not required for ribosome release by RRF in translational coupling. The data are consistent with the interpretation that RRF functions mostly as a ribosome releasing factor rather than ribosome splitting factor. Additionally, the in vivo studies showed that short (less than 5 codons) u-ORF inhibited d-ORF reading by ribosomes finishing u-ORF reading, suggesting that the termination process in short ORF is not similar to that in normal ORF. This means that all the preexisting studies on RRF with short mRNA may not represent what goes on in natural termination step.

Targeting the αVß3/NgR2 pathway in neuroendocrine prostate cancer.

Highly aggressive, metastatic, neuroendocrine prostate cancer, which typically develops from prostate cancer cells acquiring resistance to androgen deprivation therapy, is associated with limited treatment options and hence poor prognosis. We have previously demonstrated that the αVß3 integrin is over-expressed in neuroendocrine prostate cancer. We now show that LM609, a monoclonal antibody that specifically targets the human αVß3 integrin, hinders the growth of neuroendocrine prostate cancer patient-derived xenografts in vivo. Our group has recently identified a novel αVß3 integrin binding partner, NgR2, responsible for regulating the expression of neuroendocrine markers and for inducing neuroendocrine differentiation in prostate cancer cells. Through in vitro functional assays, we here demonstrate that NgR2 is crucial in promoting cell adhesion to αVß3 ligands. Moreover, we describe for the first time co-fractionation of αVß3 integrin and NgR2 in small extracellular vesicles derived from metastatic prostate cancer patients' plasma. These prostate cancer patient-derived small extracellular vesicles have a functional impact on human monocytes, increasing their adhesion to fibronectin. The monocytes incubated with small extracellular vesicles do not show an associated change in conventional polarization marker expression and appear to be in an early stage that may be defined as "adhesion competent". Overall, these findings allow us to better understand integrin-directed signaling and cell-cell communication during cancer progression. Furthermore, our results pave the way for new diagnostic and therapeutic perspectives for patients affected by neuroendocrine prostate cancer.

Efficient Modeling and Simulation of PMUT Arrays in Various Ambients.

This paper presents a numerical reduced-order modeling (ROM) approach for complex multi-layered arrays of piezoelectric micromachined ultrasonic transducers (PMUTs). The numerical modeling technique adopted to generate an array of PMUTs consisting of a considerable number of transducers allows for a large reduction in computational cost without reducing accuracy. The modeling idea is based on coupling shell elements applied to the PMUT structural layers with 3D-solid elements applied to the piezoelectric layer. A set of eigenfrequency and frequency domain analyses are presented considering a single ROM of a PMUT performing in different ambients and the performing central frequencies are obtained for every considered scenario. A unique arrangement of 228 PMUTs is presented and tested for its ability to transmit and receive acoustic waves. The operating frequency band of the array and the level of interference and cross-talk among different PMUTs in the near field are estimated. Finally, the results from a preliminary experimental test performed to analyze the acoustic abilities of an 8 × 8 array of PMUTs are presented. A corresponding numerical model is created and the obtained results matched the experimental data, leading to a validation of the modeling technique proposed in this work.

Small extracellular vesicle-mediated ITGB6 siRNA delivery downregulates the αVß6 integrin and inhibits adhesion and migration of recipient prostate cancer cells.

The αVß6 integrin, an epithelial-specific cell surface receptor absent in normal prostate and expressed during prostate cancer (PrCa) progression, is a therapeutic target in many cancers. Here, we report that transcript levels of ITGB6 (encoding the ß6 integrin subunit) are significantly increased in metastatic castrate-resistant androgen receptor-negative prostate tumors compared to androgen receptor-positive prostate tumors. In addition, the αVß6 integrin protein levels are significantly elevated in androgen receptor-negative PrCa patient derived xenografts (PDXs) compared to androgen receptor-positive PDXs. In vitro, the androgen receptor-negative PrCa cells express high levels of the αVß6 integrin compared to androgen receptor-positive PrCa cells. Additionally, expression of androgen receptor (wild type or variant 7) in androgen receptor-negative PrCa cells downregulates the expression of the ß6 but not αV subunit compared to control cells. We demonstrate an efficient strategy to therapeutically target the αVß6 integrin during PrCa progression by using short interfering RNA (siRNA) loaded into PrCa cell-derived small extracellular vesicles (sEVs). We first demonstrate that fluorescently-labeled siRNAs can be efficiently loaded into PrCa cell-derived sEVs by electroporation. By confocal microscopy, we show efficient internalization of these siRNA-loaded sEVs into PrCa cells. We show that sEV-mediated delivery of ITGB6-targeting siRNAs into PC3 cells specifically downregulates expression of the ß6 subunit. Furthermore, treatment with sEVs encapsulating ITGB6 siRNA significantly reduces cell adhesion and migration of PrCa cells on an αVß6-specific substrate, LAP-TGFß1. Our results demonstrate an approach for specific targeting of the αVß6 integrin in PrCa cells using sEVs encapsulating ITGB6-specific siRNAs.

STEAP1-4 (Six-Transmembrane Epithelial Antigen of the Prostate 1-4) and Their Clinical Implications for Prostate Cancer.

Six-Transmembrane Epithelial Antigen of the Prostate 1-4 (STEAP1-4) compose a family of metalloproteinases involved in iron and copper homeostasis and other cellular processes. Thus far, five homologs are known: STEAP1, STEAP1B, STEAP2, STEAP3, and STEAP4. In prostate cancer, STEAP1, STEAP2, and STEAP4 are overexpressed, while STEAP3 expression is downregulated. Although the metalloreductase activities of STEAP1-4 are well documented, their other biological functions are not. Furthermore, the properties and expression levels of STEAP heterotrimers, homotrimers, heterodimers, and homodimers are not well understood. Nevertheless, studies over the last few decades have provided sufficient impetus to investigate STEAP1-4 as potential biomarkers and therapeutic targets for prostate cancer. In particular, STEAP1 is the target of many emerging immunotherapies. Herein, we give an overview of the structure, physiology, and pathophysiology of STEAP1-4 to provide context for past and current efforts to translate STEAP1-4 into the clinic.

The NOGO receptor NgR2, a novel αVß3 integrin effector, induces neuroendocrine differentiation in prostate cancer.

Androgen deprivation therapies aimed to target prostate cancer (PrCa) are only partially successful given the occurrence of neuroendocrine PrCa (NEPrCa), a highly aggressive and highly metastatic form of PrCa, for which there is no effective therapeutic approach. Our group has demonstrated that while absent in prostate adenocarcinoma, the αVß3 integrin expression is increased during PrCa progression toward NEPrCa. Here, we show a novel pathway activated by αVß3 that promotes NE differentiation (NED). This novel pathway requires the expression of a GPI-linked surface molecule, NgR2, also known as Nogo-66 receptor homolog 1. We show here that NgR2 is upregulated by αVß3, to which it associates; we also show that it promotes NED and anchorage-independent growth, as well as a motile phenotype of PrCa cells. Given our observations that high levels of αVß3 and, as shown here, of NgR2 are detected in human and mouse NEPrCa, our findings appear to be highly relevant to this aggressive and metastatic subtype of PrCa. This study is novel because NgR2 role has only minimally been investigated in cancer and has instead predominantly been analyzed in neurons. These data thus pave new avenues toward a comprehensive mechanistic understanding of integrin-directed signaling during PrCa progression toward a NE phenotype.

Differential expression of αVß3 and αVß6 integrins in prostate cancer progression.

Neuroendocrine prostate cancer (NEPrCa) arises de novo or after accumulation of genomic alterations in pre-existing adenocarcinoma tumors in response to androgen deprivation therapies. We have provided evidence that small extracellular vesicles released by PrCa cells and containing the αVß3 integrin promote neuroendocrine differentiation of PrCa in vivo and in vitro. Here, we examined αVß3 integrin expression in three murine models carrying a deletion of PTEN (SKO), PTEN and RB1 (DKO), or PTEN, RB1 and TRP53 (TKO) genes in the prostatic epithelium; of these three models, the DKO and TKO tumors develop NEPrCa with a gene signature comparable to those of human NEPrCa. Immunostaining analysis of SKO, DKO and TKO tumors shows that αVß3 integrin expression is increased in DKO and TKO primary tumors and metastatic lesions, but absent in SKO primary tumors. On the other hand, SKO tumors show higher levels of a different αV integrin, αVß6, as compared to DKO and TKO tumors. These results are confirmed by RNA-sequencing analysis. Moreover, TRAMP mice, which carry NEPrCa and adenocarcinoma of the prostate, also have increased levels of αVß3 in their NEPrCa primary tumors. In contrast, the αVß6 integrin is only detectable in the adenocarcinoma areas. Finally, analysis of 42 LuCaP patient-derived xenografts and primary adenocarcinoma samples shows a positive correlation between αVß3, but not αVß6, and the neuronal marker synaptophysin; it also demonstrates that αVß3 is absent in prostatic adenocarcinomas. In summary, we demonstrate that αVß3 integrin is upregulated in NEPrCa primary and metastatic lesions; in contrast, the αVß6 integrin is confined to adenocarcinoma of the prostate. Our findings suggest that the αVß3 integrin, but not αVß6, may promote a shift in lineage plasticity towards a NE phenotype and might serve as an informative biomarker for the early detection of NE differentiation in prostate cancer.

On the Effects of Package on the PMUTs Performances-Multiphysics Model and Frequency Analyses.

This paper deals with a multiphysics numerical modelling via finite element method (FEM) of an air-coupled array piezoelectric micromachined ultrasonic transducers (PMUTs). The proposed numerical model is fully 3D with the following features: the presence of the fabrication induced residual stresses, which determine a geometrically non-linear initial deformed configuration of the diaphragms and a remarkable shift of the fundamental frequency; the multiple coupling between different physics, namely electro-mechanical-coupling for the piezo-electric model, acoustic-structure interaction at the acoustic-structure interface and pressure acoustics in the surrounding air. The model takes into account the complete set of PMUTs belonging to the silicon die in a 4 x 4 array configuration and the protective package, as well. The results have been validated by experimental data, in terms of initial static pre-deflected configuration of the diaphragms and frequency response function of the PMUT. The numerical procedure was applied, to analyze different package configurations of the device, to study the influence of the holes on the acoustic transmission in terms of SPL and propagation pattern and consequently extract a set of design guidelines.

Methods for extracellular vesicle isolation from cancer cells.

Cells are known to release different types of vesicles such as small extracellular vesicles (sEVs) and large extracellular vesicles (LEVs). sEVs and LEVs play important roles in intercellular communication, pre-metastatic niche formation, and disease progression; both can be detected cell culture media and biological fluids. sEVs and LEVs contain a variety of protein and RNA cargo, and they are believed to impact many biological functions of the recipient cells upon their internalization or binding to cell surface proteins. It has recently been established that standard isolation techniques, such as differential ultracentrifugation, yield a mixed population of EVs. However, density gradient ultracentrifugation has been reported to allow the isolation of sEVs without cellular debris. Here, we describe the most common methods used to isolate sEVs from cell culture medium, mouse and human plasma, and a new technique for isolating sEVs from tissues as well. This article also provides detailed procedures to isolate LEVs.

Small extracellular vesicles modulated by αVß3 integrin induce neuroendocrine differentiation in recipient cancer cells.

The ability of small extracellular vesicles (sEVs) to reprogram cancer cells is well established. However, the specific sEV components able to mediate aberrant effects in cancer cells have not been characterized. Integrins are major players in mediating sEV functions. We have previously reported that the αVß3 integrin is detected in sEVs of prostate cancer (PrCa) cells and transferred into recipient cells. Here, we investigate whether sEVs from αVß3-expressing cells affect tumour growth differently than sEVs from control cells that do not express αVß3. We compared the ability of sEVs to stimulate tumour growth, using sEVs isolated from PrCa C4-2B cells by iodixanol density gradient and characterized with immunoblotting, nanoparticle tracking analysis, immunocapturing and single vesicle analysis. We incubated PrCa cells with sEVs and injected them subcutaneously into nude mice to measure in vivo tumour growth or analysed in vitro their anchorage-independent growth. Our results demonstrate that a single treatment with sEVs shed from C4-2B cells that express αVß3, but not from control cells, stimulates tumour growth and induces differentiation of PrCa cells towards a neuroendocrine phenotype, as quantified by increased levels of neuroendocrine markers. In conclusion, the expression of αVß3 integrin generates sEVs capable of reprogramming cells towards an aggressive phenotype.

The αvß6 integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis.

Prostate cancer (PrCa) cells crosstalk with the tumour microenvironment by releasing small extracellular vesicles (sEVs). sEVs, as well as large extracellular vesicles (LEVs), isolated via iodixanol density gradients from PrCa cell culture media, express the epithelial-specific αvß6 integrin, which is known to be induced in cancer. In this study, we show sEV-mediated protein transfer of αvß6 integrin to microvascular endothelial cells (human microvascular endothelial cells 1 - HMEC1) and demonstrate that de novo αvß6 integrin expression is not caused by increased mRNA levels. Incubation of HMEC1 with sEVs isolated from PrCa PC3 cells that express the αvß6 integrin results in a highly significant increase in the number of nodes, junctions and tubules. In contrast, incubation of HMEC1 with sEVs isolated from ß6 negative PC3 cells, generated by shRNA against ß6, results in a reduction in the number of nodes, junctions and tubules, a decrease in survivin levels and an increase in a negative regulator of angiogenesis, pSTAT1. Furthermore, treatment of HMEC1 with sEVs generated by CRISPR/Cas9-mediated down-regulation of ß6, causes up-regulation of pSTAT1. Overall, our findings suggest that αvß6 integrin in cancer sEVs regulates angiogenesis during PrCa progression.

A Comparative Analysis of CMUT Receiving Architectures for the Design Optimization of Integrated Transceiver Front Ends.

A formal comparison between fundamental RX amplifier configurations for capacitive micromachined ultrasonic transducers (CMUTs) is proposed in this paper. The impact on both RX and the pulse-echo frequency response and on the output SNR is thoroughly analyzed and discussed. It is shown that the resistive-feedback amplifier yields a bandpass RX frequency response, while both open-loop voltage and capacitive-feedback amplifiers exhibit a low-pass frequency response. For a given power dissipation, it is formally proved that a capacitive-feedback amplifier provides a remarkable SNR improvement against the commonly adopted resistive feedback stage, achieved at the expense of a reduced pulse-echo center frequency, making its use convenient in low-frequency and midfrequency ultrasound imaging applications. The advantage mostly comes from a much lower noise contributed by the active devices, especially with low- Q , broadband transducers. The results of the analysis are applied to the design of a CMUT front end in BIPOLAR-CMOS-DMOS Silicon-on-Insulator technology operating at 10-MHz center frequency. It comprises a low-power RX amplifier, a high-voltage Transmission/Reception switch, and a 100-V TX driver. Extensive electrical characterization, pulse-echo measurements, and imaging results are shown. Compared with previously reported CMUT front ends, this transceiver demonstrates the highest dynamic range and state-of-the-art noise performance with an RX amplifier power dissipation of 1 mW.

An Experimental Protocol for Assessing the Performance of New Ultrasound Probes Based on CMUT Technology in Application to Brain Imaging.

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ultrasound (US) probes. Particularly, a more realistic analysis with application-specific imaging targets can be extremely valuable to assess the expected performance of US probes in their potential clinical field of application. The experimental protocol presented in this work was purposely designed to provide an application-specific assessment procedure for newly-developed US probe prototypes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology in relation to brain imaging. The protocol combines the use of a bovine brain fixed in formalin as the imaging target, which ensures both realism and repeatability of the described procedures, and of neuronavigation techniques borrowed from neurosurgery. The US probe is in fact connected to a motion tracking system which acquires position data and enables the superposition of US images to reference Magnetic Resonance (MR) images of the brain. This provides a means for human experts to perform a visual qualitative assessment of the US probe imaging performance and to compare acquisitions made with different probes. Moreover, the protocol relies on the use of a complete and open research and development system for US image acquisition, i.e. the Ultrasound Advanced Open Platform (ULA-OP) scanner. The manuscript describes in detail the instruments and procedures involved in the protocol, in particular for the calibration, image acquisition and registration of US and MR images. The obtained results prove the effectiveness of the overall protocol presented, which is entirely open (within the limits of the instrumentation involved), repeatable, and covers the entire set of acquisition and processing activities for US images.

Chemical and structural characterization of a model Post-Termination Complex (PoTC) for the ribosome recycling reaction: Evidence for the release of the mRNA by RRF and EF-G.

A model Post-Termination Complex (PoTC) used for the discovery of Ribosome Recycling Factor (RRF) was purified and characterized by cryo-electron microscopic analysis and biochemical methods. We established that the model PoTC has mostly one tRNA, at the P/E or P/P position, together with one mRNA. The structural studies were supported by the biochemical measurement of bound tRNA and mRNA. Using this substrate, we establish that the release of tRNA, release of mRNA and splitting of ribosomal subunits occur during the recycling reaction. Order of these events is tRNA release first followed by mRNA release and splitting almost simultaneously. Moreover, we demonstrate that IF3 is not involved in any of the recycling reactions but simply prevents the re-association of split ribosomal subunits. Our finding demonstrates that the important function of RRF includes the release of mRNA, which is often missed by the use of a short ORF with the Shine-Dalgarno sequence near the termination site.

A volumetric CMUT-based ultrasound imaging system simulator with integrated reception and µ-beamforming electronics models.

In modern ultrasound imaging devices, two-dimensional probes and electronic scanning allow volumetric imaging of anatomical structures. When dealing with the design of such complex 3-D ultrasound (US) systems, as the number of transducers and channels dramatically increases, new challenges concerning the integration of electronics and the implementation of smart micro-beamforming strategies arise. Hence, the possibility to predict the behavior of the whole system is mandatory. In this paper, we propose and describe an advanced simulation tool for ultrasound system modeling and simulation, which conjugates the US propagation and scattering, signal transduction, electronic signal conditioning, and beamforming in a single environment. In particular, we present the architecture and model of an existing 16-channel integrated receiver, which includes an amplification and micro-beamforming stage, and validate it by comparison with circuit simulations. The developed model is then used in conjunction with the transducer and US field models to perform a system simulation, aimed at estimating the performance of an example 3-D US imaging system that uses a capacitive micromachined ultrasonic transducer (CMUT) 2-D phased-array coupled to the modeled reception front-end. Results of point spread function (PSF) calculations, as well as synthetic imaging of a virtual phantom, show that this tool is actually able to model the complete US image reconstruction process, and that it could be used to quickly provide valuable system-level feedback for an optimized tuning of electronic design parameters.

Structural and functional characterization of the bacterial translocation inhibitor GE82832.

The structure of GE82832, a translocation inhibitor produced by a soil microorganism, is shown to be highly related to that of dityromycin, a bicyclodecadepsipeptide antibiotic discovered long ago whose characterization had never been pursued beyond its structural elucidation. GE82832 and dityromycin were shown to interfere with both aminoacyl-tRNA and mRNA movement and with the Pi release occurring after ribosome- and EF-G-dependent GTP hydrolysis. These findings and the unusual ribosomal localization of GE82832/dityromycin near protein S13 suggest that the mechanism of inhibition entails an interference with the rotation of the 30S subunit "head" which accompanies the ribosome-unlocking step of translocation.

Simulating ultrasound fields for 2D phased-array probes design optimization.

Nowadays, ultrasound diagnostic imaging is one of the non-invasive techniques mostly used in the clinical practice. Recent advances in this field have brought to the development of small and portable systems. New bidimensional probes consisting of 2D phased arrays, allow to obtain real-time 3D representations of moving organs and blood vessels anatomy. Being the complexity of such 4D ultrasound imaging systems significantly increased, new challenges concerning electronics integration arise for designers. In this paper a software simulator is described, which has been developed in order to model ultrasound wave generation, pressure field distribution and echoes reception, with the aim to become a useful tool for optimizing the probe design. The paper mainly focuses on linear ultrasound field modeling; preliminary results on non-linear interactions with contrast agents are also here introduced.