Time-resolved proximity proteomics uncovers a membrane tension-sensitive caveolin-1 interactome at the rear of migrating cells.

Caveolae are small membrane pits with fundamental roles in mechanotransduction. Several studies have shown that caveolae flatten out in response to increased membrane tension, thereby acting as a mechanosensitive membrane reservoir that buffers acute mechanical stress. Caveolae have also been implicated in the control of RhoA/ROCK-mediated actomyosin contractility at the rear of migrating cells. However, how membrane tension controls the organisation of caveolae and their role in mechanotransduction remains unclear. To address this, we systematically quantified protein-protein interactions of caveolin-1 in migrating RPE1 cells at steady state and in response to an acute increase in membrane tension using biotin-based proximity labelling and quantitative mass spectrometry. Our data show that caveolae are highly enriched at the rear of migrating RPE1 cells and that membrane tension rapidly and reversibly disrupts the caveolar protein coat. Membrane tension also detaches caveolin-1 from focal adhesion proteins and several mechanosensitive regulators of cortical actin including filamins and cortactin. In addition, we present evidence that ROCK and the RhoGAP ARHGAP29 associate with caveolin-1 in a manner dependent on membrane tension, with ARHGAP29 influencing caveolin-1 Y14 phosphorylation, caveolae rear localisation, and RPE1 cell migration. Taken together, our work uncovers a membrane tension-sensitive coupling between caveolae and the rear-localised F-actin cytoskeleton. This provides a framework for dissecting the molecular mechanisms underlying caveolae-regulated mechanotransduction pathways.

Strategies and safety simulations for ultrasonic cervical spinal cord neuromodulation.

Objective. Focused ultrasound spinal cord neuromodulation has been demonstrated in small animals. However, most of the tested neuromodulatory exposures are similar in intensity and exposure duration to the reported small animal threshold for possible spinal cord damage. All efforts must be made to minimize the risk and assure the safety of potential human studies, while maximizing potential treatment efficacy. This requires an understanding of ultrasound propagation and heat deposition within the human spine.Approach. Combined acoustic and thermal modelling was used to assess the pressure and heat distributions produced by a 500 kHz source focused to the C5/C6 level via two approaches (a) the posterior acoustic window between vertebral posterior arches, and (b) the lateral intervertebral foramen from which the C6 spinal nerve exits. Pulse trains of fifty 0.1 s pulses (pulse repetition frequency: 0.33 Hz, free-field spatial peak pulse-averaged intensity: 10 W cm-2) were simulated for four subjects and for ±10 mm translational and ±10∘rotational source positioning errors.Main results.Target pressures ranged between 20%-70% of free-field spatial peak pressures with the posterior approach, and 20%-100% with the lateral approach. When the posterior source was optimally positioned, peak spine heating values were below 1 ∘C, but source mispositioning resulted in bone heating up to 4 ∘C. Heating with the lateral approach did not exceed 2 ∘C within the mispositioning range. There were substantial inter-subject differences in target pressures and peak heating values. Target pressure varied three to four-fold between subjects, depending on approach, while peak heating varied approximately two-fold between subjects. This results in a nearly ten-fold range between subjects in the target pressure achieved per degree of maximum heating.Significance. This study highlights the utility of trans-spine ultrasound simulation software and need for precise source-anatomy positioning to assure the subject-specific safety and efficacy of focused ultrasound spinal cord therapies.

ITRUSST consensus on standardised reporting for transcranial ultrasound stimulation.

As transcranial ultrasound stimulation (TUS) advances as a precise, non-invasive neuromodulatory method, there is a need for consistent reporting standards to enable comparison and reproducibility across studies. To this end, the International Transcranial Ultrasonic Stimulation Safety and Standards Consortium (ITRUSST) formed a subcommittee of experts across several domains to review and suggest standardised reporting parameters for low intensity TUS, resulting in the guide presented here. The scope of the guide is limited to reporting the ultrasound aspects of a study. The guide and supplementary material provide a simple checklist covering the reporting of: (1) the transducer and drive system, (2) the drive system settings, (3) the free field acoustic parameters, (4) the pulse timing parameters, (5) in situ estimates of exposure parameters in the brain, and (6) intensity parameters. Detailed explanations for each of the parameters, including discussions on assumptions, measurements, and calculations, are also provided.

Review of the Low-Temperature Acoustic Properties of Water, Aqueous Solutions, Lipids, and Soft Biological Tissues.

Existing data on the acoustic properties of low-temperature biological materials is limited and widely dispersed across fields. This makes it difficult to employ this information in the development of ultrasound applications in the medical field, such as cryosurgery and rewarming of cryopreserved tissues. In this review, the low-temperature acoustic properties of biological materials, and the measurement methods used to acquire them were collected from a range of scientific fields. The measurements were reviewed from the acoustic setup to thermal methodologies for samples preparation, temperature monitoring, and system insulation. The collected data contain the longitudinal and shear velocity, and attenuation coefficient of biological soft tissues and biologically relevant substances-water, aqueous solutions, and lipids-in the temperature range down to -50 °C and in the frequency range from 108 kHz to 25 MHz. The multiple reflection method (MRM) was found to be the preferred method for low-temperature samples, with a buffer rod inserted between the transducer and sample to avoid direct contact. Longitudinal velocity changes are observed through the phase transition zone, which is sharp in pure water, and occurs more slowly and at lower temperatures with added solutes. Lipids show longer transition zones with smaller sound velocity changes; with the longitudinal velocity changes observed during phase transition in tissues lying between these two extremes. More general conclusions on the shear velocity and attenuation coefficient at low-temperatures are restricted by the limited data. This review enhance knowledge guiding for further development of ultrasound applications in low-temperature biomedical fields, and may help to increase the precision and standardization of low-temperature acoustic property measurements.

ITRUSST Consensus on Standardised Reporting for Transcranial Ultrasound Stimulation.

As transcranial ultrasound stimulation (TUS) advances as a precise, non-invasive neuromodulatory method, there is a need for consistent reporting standards to enable comparison and reproducibility across studies. To this end, the International Transcranial Ultrasonic Stimulation Safety and Standards Consortium (ITRUSST) formed a subcommittee of experts across several domains to review and suggest standardised reporting parameters for low intensity TUS, resulting in the guide presented here. The scope of the guide is limited to reporting the ultrasound aspects of a study. The guide and supplementary material provide a simple checklist covering the reporting of: (1) the transducer and drive system, (2) the drive system settings, (3) the free field acoustic parameters, (4) the pulse timing parameters, (5) in situ estimates of exposure parameters in the brain, and (6) intensity parameters. Detailed explanations for each of the parameters, including discussions on assumptions, measurements, and calculations, are also provided.

Safety Review of Therapeutic Ultrasound for Spinal Cord Neuromodulation and Blood-Spinal Cord Barrier Opening.

New focused ultrasound spinal cord applications have emerged, particularly those improving therapeutic agent delivery to the spinal cord via blood-spinal cord barrier opening and the neuromodulation of spinal cord tracts. One hurdle in the development of these applications is safety. It may be possible to use safety trends from seminal and subsequent works in focused ultrasound to guide the development of safety guidelines for spinal cord applications. We collated data from decades of pre-clinical studies and illustrate a clear relationship between damage, time-averaged spatial peak intensity and exposure duration. This relationship suggests a thermal mechanism underlies ultrasound-induced spinal cord damage. We developed minimum and mean thresholds for damage from these pre-clinical studies. When these thresholds were plotted against the parameters used in recent pre-clinical ultrasonic spinal cord neuromodulation studies, the majority of the neuromodulation studies were near or above the minimum threshold. This suggests that a thermal neuromodulatory effect may exist for ultrasonic spinal cord neuromodulation, and that the thermal dose must be carefully controlled to avoid damage to the spinal cord. By contrast, the intensity-exposure duration threshold had no predictive value when applied to blood-spinal cord barrier opening studies that employed injected contrast agents. Most blood-spinal cord barrier opening studies observed slight to severe damage, except for small animal studies that employed an active feedback control method to limit pressures based on measured bubble oscillation behavior. The development of new focused ultrasound spinal cord applications perhaps reflects the recent success in the development of focused ultrasound brain applications, and recent work has begun on the translation of these technologies from brain to spinal cord. However, a great deal of work remains to be done, particularly with respect to developing and accepting safety standards for these applications.

Transcranial focused ultrasound-mediated neurochemical and functional connectivity changes in deep cortical regions in humans.

Low-intensity transcranial ultrasound stimulation (TUS) is an emerging non-invasive technique for focally modulating human brain function. The mechanisms and neurochemical substrates underlying TUS neuromodulation in humans and how these relate to excitation and inhibition are still poorly understood. In 24 healthy controls, we separately stimulated two deep cortical regions and investigated the effects of theta-burst TUS, a protocol shown to increase corticospinal excitability, on the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and functional connectivity. We show that theta-burst TUS in humans selectively reduces GABA levels in the posterior cingulate, but not the dorsal anterior cingulate cortex. Functional connectivity increased following TUS in both regions. Our findings suggest that TUS changes overall excitability by reducing GABAergic inhibition and that changes in TUS-mediated neuroplasticity last at least 50 mins after stimulation. The difference in TUS effects on the posterior and anterior cingulate could suggest state- or location-dependency of the TUS effect-both mechanisms increasingly recognized to influence the brain's response to neuromodulation.

The Efficacy of Text-Based Mentoring for Postpartum Mothers: A Pilot Study.

OBJECTIVES: Technology-based outreach offers promise for providing support to a broad population of postpartum mothers while keeping costs low. However, research on the efficacy of this approach is scarce. We conducted a pre-registered randomized pilot trial of the effects of a novel technology-based approach for supporting postpartum mothers - via text-based mentoring - from infant's birth through 18 months. METHODS: Mothers (n = 201) were recruited at West Penn Hospital in Pittsburgh, PA in the days immediately following delivery. Treatment mothers were matched with volunteer mentors who communicated with them entirely via text messages. Control mothers received monthly one-way texts on basic safety topics. Measures were collected via hospital records and mother surveys. We estimated treatment effects on mothers' parenting stress, mental health, knowledge of child development, engagement in language and literacy activities, and child milestones at 4- and 18-months postpartum. We used a systematic coding approach and simple descriptive statistics to analyze the treatment mother-mentor texting transcripts. RESULTS: We found no statistically significant impacts on targeted outcomes. However, impacts for some outcomes were meaningfully large (> 0.2 SDs). Analyses of texting transcripts showed that most mothers stayed engaged for the full 18-month study period and that mother-mentor pairs primarily discussed maternal wellbeing and child-focused topics. CONCLUSIONS FOR PRACTICE: Postpartum mothers will engage with mentors in a text-based mentoring program around important maternal and child health topics. More research and development on technology-based supports for parents in the early childhood years is needed.

Fiber-optic hydrophone for detection of high-intensity ultrasound waves.

Fiber-optic hydrophones (FOHs) are widely used to detect high-intensity focused ultrasound (HIFU) fields. The most common type consists of an uncoated single-mode fiber with a perpendicularly cleaved end face. The main disadvantage of these hydrophones is their low signal-to-noise ratio (SNR). To increase the SNR, signal averaging is performed, but the associated increased acquisition times hinder ultrasound field scans. In this study, with a view to increasing SNR while withstanding HIFU pressures, the bare FOH paradigm is extended to include a partially reflective coating on the fiber end face. Here, a numerical model based on the general transfer-matrix method was implemented. Based on the simulation results, a single-layer, 172 nm TiO2-coated FOH was fabricated. The frequency range of the hydrophone was verified from 1 to 30 MHz. The SNR of the acoustic measurement with the coated sensor was 21 dB higher than that of the uncoated one. The coated sensor successfully withstood a peak positive pressure of 35 MPa for 6000 pulses.

open-UST: An Open-Source Ultrasound Tomography Transducer Array System.

Fast imaging methods are needed to promote clinical adoption of ultrasound tomography (UST), and more widely available UST hardware could support the experimental validation of new measurement configurations. In this work, an open-source 256-element transducer ring array was developed (morganjroberts.github. io/open-UST) and manufactured using rapid prototyping, for only £2k. Novel manufacturing techniques were used, resulting in a 1.17° mean beam axis skew angle, a [Formula: see text] mean element position error, and a [Formula: see text] deviation in matching layer thickness. The nominal acoustic performance was measured using hydrophone scans and watershot data, and the 61.2 dB signal-to-noise ratio (SNR), 55.4° opening angle, 10.2 mm beamwidth, and 54% transmit-receive bandwidth (-12 dB) were found to be similar to existing systems and compatible with state-of-the-art full-waveform-inversion image reconstruction methods. The interelement variation in acoustic performance was typically < 10% without using normalization, meaning that the elements can be modeled identically during image reconstruction, removing the need for individual source definitions based on hydrophone measurements. Finally, data from a phantom experiment were successfully reconstructed. These results demonstrate that the open-UST system is accessible for users and is suitable for UST imaging research.

Experiments and simulations demonstrating the rapid ultrasonic rewarming of frozen tissue cryovials.

The development of methods to safely rewarm large cryopreserved biological samples remains a barrier to the widespread adoption of cryopreservation. Here, experiments and simulations were performed to demonstrate that ultrasound can increase rewarming rates relative to thermal conduction alone. An ultrasonic rewarming setup based on a custom 444 kHz tubular piezoelectric transducer was designed, characterized, and tested with 2 ml cryovials filled with frozen ground beef. Rewarming rates were characterized in the -20 °C to 5 °C range. Thermal conduction-based rewarming was compared to thermal conduction plus ultrasonic rewarming, demonstrating a tenfold increase in rewarming rate when ultrasound was applied. The maximum recorded rewarming rate with ultrasound was 57° C/min, approximately 2.5 times faster than with thermal conduction alone. Coupled acoustic and thermal simulations were developed and showed good agreement with the heating rates demonstrated experimentally and were also used to demonstrate spatial heating distributions with small (<3° C) temperature differentials throughout the sample when the sample was below 0° C. The experiments and simulations demonstrate the potential for ultrasonic cryovial rewarming with a possible application to large volume rewarming, as faster rewarming rates may improve the viability of cryopreserved tissues and reduce the time needed for cells to regain normal function.

Measurement and simulation of steered acoustic fields generated by a multielement array for therapeutic ultrasound.

Modelling of fields generated by therapeutic ultrasound arrays can be prone to errors arising from differences from nominal transducer parameters, and variations in relative outputs of array elements when driven under different conditions, especially when simulating steered fields. Here, the effect of element size, element positions, relative source pressure variations, and electrical crosstalk on the accuracy of modelling pressure fields generated by a 555 kHz 32-element ultrasonic array were investigated. For this transducer, errors in pressure amplitude and focal position were respectively reduced from 20% to 4% and 3.3 mm to 1.5 mm using crosstalk prediction, and experimentally determined positions.

Prostatic calcifications: Quantifying occurrence, radiodensity, and spatial distribution in prostate cancer patients.

BACKGROUND: To evaluate the prevalence, density, and distribution of prostate calcification in patients with prostate cancer. METHODS: Patients who underwent both Gallium-68 PSMA PET/CT and MRI of the prostate over the course of a year were selected for analysis. The CT images with visible calcifications within the prostate were included and calcifications automatically isolated using a threshold of 130 HU. The corresponding multiparametric MRI was assessed and the peripheral zone, transition zone, MRI-visible tumor, and urethra manually contoured. The contoured MRI and CT images were registered using rigid registration, and calcifications mapped automatically to the MRI contours. RESULTS: A total of 85 men (age range 50-88, mean 69 years, standard deviation 7.2 years) were assessed. The mean serum Prostate Specific Antigen PSA was 16.7, range 0.12 to 94.4. Most patients had intermediate-risk disease (68%; Gleason grade group 2 and 3), 26% had high-risk disease (Gleason grade group 4 and 5), and 6% had low-risk disease (Gleason grade group 1). Forty-six patients out of 85 (54%) had intraprostatic calcification. Calcification occurred more in transition zone than the peripheral zone (65% vs. 35%). The mean density of the calcification was 227 HU (min 133, max 1,966 HU). In 12 patients, the calcification was within an MRI-visible tumor, in 24 patients, there were calcifications within a 9 mm distance of the tumor border, and in 9 patients, there were calcifications located between the urethra and tumor. CONCLUSIONS: Calcifications are common in patients with prostate cancer. Their density and location may make them a significant consideration when planning treatment or retreatment with some types of minimally invasive therapy.

New insights into the organization and regulation of the apical polarity network in mammalian epithelial cells.

Cell polarity is a fundamental property of most animal cells and is critical during development and for most cell and tissue functions. Epithelial cells are organized into apical and basolateral compartments, and this intrinsic cellular asymmetry is essential for all functions that are carried out by epithelial tissue. The establishment of a polarized epithelial phenotype is orchestrated by major rearrangements of the cell cytoskeleton, polarized membrane trafficking, the formation and maturation of epithelial cell junctions, cell signaling pathways, and the generation of cortical phospholipid asymmetry. These processes need to be coordinated precisely in time and space and integrated with physical and chemical signals from the environment, failure of which leads to severe developmental disorders and various human diseases. At the heart of this regulatory network are the evolutionarily conserved polarity modules Par, Crumbs, and Scribble, whose components engage in complex cooperative and antagonistic interactions to compartmentalize and functionalize the epithelial cell cortex and to control the spatiotemporal activity of downstream polarity effectors. In this review, we will discuss recent insights into the organization and regulation of the mammalian Par and Crumbs modules and outline a hypothetical framework of how these proteins orchestrate epithelial polarity development, HIPPO signaling, and actomyosin activity at the apical-lateral border.

In vivo crystals reveal critical features of the interaction between cystic fibrosis transmembrane conductance regulator (CFTR) and the PDZ2 domain of Na+/H+ exchange cofactor NHERF1.

Crystallization of recombinant proteins has been fundamental to our understanding of protein function, dysfunction, and molecular recognition. However, this information has often been gleaned under extremely nonphysiological protein, salt, and H+ concentrations. Here, we describe the development of a robust Inka1-Box (iBox)-PAK4cat system that spontaneously crystallizes in several mammalian cell types. The semi-quantitative assay described here allows the measurement of in vivo protein-protein interactions using a novel GFP-linked reporter system that produces fluorescent readouts from protein crystals. We combined this assay with in vitro X-ray crystallography and molecular dynamics studies to characterize the molecular determinants of the interaction between the PDZ2 domain of Na+/H+ exchange regulatory cofactor NHE-RF1 (NHERF1) and cystic fibrosis transmembrane conductance regulator (CFTR), a protein complex pertinent to the genetic disease cystic fibrosis. These experiments revealed the crystal structure of the extended PDZ domain of NHERF1 and indicated, contrary to what has been previously reported, that residue selection at positions -1 and -3 of the PDZ-binding motif influences the affinity and specificity of the NHERF1 PDZ2-CFTR interaction. Our results suggest that this system could be utilized to screen additional protein-protein interactions, provided they can be accommodated within the spacious iBox-PAK4cat lattice.

Experimental Validation of k-Wave: Nonlinear Wave Propagation in Layered, Absorbing Fluid Media.

Models of ultrasound propagation in biologically relevant media have applications in planning and verification of ultrasound therapies and computational dosimetry. To be effective, the models must be able to accurately predict both the spatial distribution and amplitude of the acoustic pressure. This requires that the models are validated in absolute terms, which for arbitrarily heterogeneous media should be performed by comparison with measurements of the acoustic field. In this article, simulations performed using the open-source k-Wave acoustics toolbox, with a measurement-based source definition, were quantitatively validated against measurements of acoustic pressure in water and layered absorbing fluid media. In water, the measured and simulated spatial-peak pressures agreed to within 3% under linear conditions and 7% under nonlinear conditions. After propagation through a planar or wedge-shaped glycerol-filled phantom, the difference in spatial-peak pressure was 8.5% and 10.7%, respectively. These differences are within or close to the expected uncertainty of the acoustic pressure measurement. The -6 dB width and length of the focus agreed to within 4% in all cases, and the focal positions were within 0.7 mm for the planar phantom and 1.2 mm for the wedge-shaped phantom. These results demonstrate that when the acoustic medium properties and geometry are well known, accurate quantitative predictions of the acoustic field can be made using k-Wave.