A Radiological Clip Design Using Ultrasound Identification to Improve Localization.

OBJECTIVE: We demonstrate the use of ultrasound to receive an acoustic signal transmitted from a radiological clip designed from a custom circuit. This signal encodes an identification number and is localized and identified wirelessly by the ultrasound imaging system. METHODS: We designed and constructed the test platform with a Teensy 4.0 microcontroller core to detect ultrasonic imaging pulses received by a transducer embedded in a phantom, which acted as the radiological clip. Ultrasound identification (USID) signals were generated and transmitted as a result. The phantom and clip were imaged using an ultrasonic array (Philips L7-4) connected to a Verasonics™ Vantage 128 system operating in pulse inversion (PI) mode. Cross-correlations were performed to localize and identify the code sequences in the PI images. RESULTS: USID signals were detected and visualized on B-mode images of the phantoms with up to sub-millimeter localization accuracy. The average detection rate across 30,400 frames of ultrasound data was 98.1%. CONCLUSION: The USID clip produced identifiable, distinguishable, and localizable signals when imaged. SIGNIFICANCE: Radiological clips are used to mark breast cancer being treated by neoadjuvant chemotherapy (NAC) via implant in or near treated lesions. As NAC progresses, available marking clips can lose visibility in ultrasound, the imaging modality of choice for monitoring NAC-treated lesions. By transmitting an active signal, more accurate and reliable ultrasound localization of these clips could be achieved and multiple clips with different ID values could be imaged in the same field of view.

In Vivo Validation of an In Situ Calibration Bead as a Reference for Backscatter Coefficient Calculation.

OBJECTIVE: The study described here was aimed at assessing the capability of quantitative ultrasound (QUS) based on the backscatter coefficient (BSC) for classifying disease states, such as breast cancer response to neoadjuvant chemotherapy and quantification of fatty liver disease. We evaluated the effectiveness of an in situ titanium (Ti) bead as a reference target in calibrating the system and mitigating attenuation and transmission loss effects on BSC estimation. METHODS: Traditional BSC estimation methods require external references for calibration, which do not account for ultrasound attenuation or transmission losses through tissues. To address this issue, we used an in situ Ti bead as a reference target, because it can be used to calibrate the system and mitigate the attenuation and transmission loss effects on estimation of the BSC. The capabilities of the in situ calibration approach were assessed by quantifying consistency of BSC estimates from rabbit mammary tumors (N = 21). Specifically, mammary tumors were grown in rabbits and when a tumor reached ≥1 cm in size, a 2 mm Ti bead was implanted in the tumor as a radiological marker and a calibration source for ultrasound. Three days later, the tumors were scanned with an L-14/5 38 array transducer connected to a SonixOne scanner with and without a slab of pork belly placed on top of the tumors. The pork belly acted as an additional source of attenuation and transmission loss. QUS parameters, specifically effective scatterer diameter (ESD) and effective acoustic concentration (EAC), were calculated using calibration spectra from both an external reference phantom and the Ti bead. RESULTS: For ESD estimation, the 95% confidence interval between measurements with and without the pork belly layer was 6.0, 27.4 using the in situ bead and 114, 135.1 with the external reference phantom. For EAC estimation, the 95% confidence intervals were -8.1, 0.5 for the bead and -41.5, -32.2 for the phantom. These results indicate that the in situ bead method has reduced bias in QUS estimates because of intervening tissue losses. CONCLUSION: The use of an in situ Ti bead as a radiological marker not only serves its traditional role but also effectively acts as a calibration target for QUS methods. This approach accounts for attenuation and transmission losses in tissue, resulting in more accurate QUS estimates and offering a promising method for enhanced disease state classification in clinical settings.

Grating lobe mitigation on large-pitch arrays using null subtraction imaging.

Null Subtraction Imaging (NSI) is a novel beamforming technique that can produce B-mode images resulting in high spatial resolution and low computational cost compared to other beamforming techniques. Previous work has demonstrated that in addition to a beam pattern with a narrow main lobe and low side lobes, NSI can also reduce or mitigate grating lobes, which can appear when the array pitch is larger than one half the wavelength of the transmitted pulse. These grating lobes can result in imaging artifacts that produce clutter and lower contrast. By lowering grating lobe levels, a larger pitch array could be used, which could allow arrays with a larger field of view while maintaining a standard element count. This could have important benefits for specific applications such as ultrasonic abdominal imaging. Experiments were conducted to examine the feasibility of using NSI with large pitch, wide field-of-view arrays. Grating lobe reduction was measured against array pitch, DC offset, and f-number. Experiments were conducted on wire targets and contrast targets in a phantom and results were further verified in vivo by imaging the liver of a rabbit. The results demonstrated that NSI was able to reduce grating lobe brightness by up to 45 dB compared to delay-and-sum (DAS) beamforming when using planewave transmissions, reduce the generalized contrast-to-noise ratio (gCNR) of grating lobe regions from 0.60 to 0.08, and maintain a similar speckle quality to DAS. The gCNR of anechoic regions also improves, increasing from 0.09 to 0.15 on an array with a pitch of 5 wavelengths. Due to significant grating lobe level reduction, NSI shows potential to improve image quality over DAS on a large pitch, wide field-of-view array.

In vivo validation of an in situ calibration bead as a reference for backscatter coefficient calculation.

Objectives: The study aims to assess the capability of Quantitative Ultrasound (QUS) based on the backscatter coefficient (BSC) for classifying disease states, such as breast cancer response to neoadjuvant chemotherapy and quantifying fatty liver disease. We evaluate the effectiveness of an in situ titanium (Ti) bead as a reference target in calibrating the system and mitigating attenuation and transmission loss effects on BSC estimation. Methods: Traditional BSC estimation methods require external references for calibration, which do not account for ultrasound attenuation or transmission losses through tissues. To address this issue, we use an in situ titanium (Ti) bead as a reference target, because it can be used to calibrate the system and mitigate the attenuation and transmission loss effects on estimation of the BSC. The capabilities of the in situ calibration approach were assessed by quantifying consistency of BSC estimates from rabbit mammary tumors (N=21). Specifically, mammary tumors were grown in rabbits and when a tumor reached 1 cm or greater in size, a 2-mm Ti bead was implanted into the tumor as a radiological marker and a calibration source for ultrasound. Three days later, the tumors were scanned with a L-14/5 38 array transducer connected to a SonixOne scanner with and without a slab of pork belly placed on top of the tumors. The pork belly acted as an additional source of attenuation and transmission loss. QUS parameters, specifically effective scatterer diameter (ESD) and effective acoustic concentration (EAC), were calculated using calibration spectra from both an external reference phantom and the Ti bead. Results: For ESD estimation, the 95% confidence interval between measurements with and without the pork belly layer was (6.0,27.4) using the in situ bead and (114, 135.1) with the external reference phantom. For EAC estimation, the 95% confidence interval were (-8.1, 0.5) for the bead and (-41.5, -32.2) for the phantom. These results indicate that the in situ bead method shows reduced bias in QUS estimates due to intervening tissue losses. Conclusions: The use of an in situ Ti bead as a radiological marker not only serves its traditional role but also effectively acts as a calibration target for QUS methods. This approach accounts for attenuation and transmission losses in tissue, resulting in more accurate QUS estimates and offering a promising method for enhanced disease state classification in clinical settings.

Detection of microcalcifications using nonlinear beamforming techniques.

OBJECTIVE: Abundant research demonstrates that early detection of cancer leads to improved patient prognoses. By detecting cancer earlier, when tumors are in their primary stages, treatment can be applied before metastases have occurred. The presence of microcalcifications (MCs) is indicative of malignancy in the breast, i.e., 30-50% of all nonpalpable breast cancers detected using mammograms are based on identifying the presence of MCs. Therefore, improving the ability to detect MCs with modern imaging technology remains an important goal. Specifically, improving the sensitivity of ultrasound imaging techniques to detect MCs in the breast will provide an important role for the early detection and diagnosis of breast cancer. METHODS: In this work, a novel nonlinear beamforming technology for ultrasonic arrays is investigated for its ability to detect MCs. The beamforming technique, called null subtraction imaging (NSI), utilizes nulls in the beam pattern to create images using ultrasound. NSI provides improved lateral resolution, a reduction in side lobes, and an accentuation of bright singular targets. Therefore, it was hypothesized that the use of NSI would result in identification of more MCs in rat tumors having a speckle background. To test this hypothesis, rats with tumors were injected with Hydroxyapatite (HA) particles to mimic MCs. Ultrasound was used to scan the rat tumors and images were constructed using conventional delay and sum and using NSI beamforming. Three readers with experience in diagnostic ultrasound imaging examined the 1,344 images and scored the presence or absence of MCs. DISCUSSION: In all, 336 different tumor image slices were recorded and each reconstructed using NSI or conventional delay and sum with Hann apodization. In every image where one or MCs were detected in the Hann reconstructions, MCs were detected in the NSI images. In nine rat tumor images, one or more MCs were detected in the NSI images but not in the Hann images. CONCLUSIONS: Statistically, the results did support the hypothesis that NSI would increase the number of MCs detected in the rat tumors.

Grating Lobe Reduction in Plane-Wave Imaging With Angular Compounding Using Subtraction of Coherent Signals.

Plane-wave imaging (PWI) with angular compounding has gained in popularity over recent years, because it provides high frame rates and good image properties. However, most linear arrays used in clinical practice have a pitch that is equal to than the wavelength of ultrasound. Hence, the presence of grating lobes is a concern for PWI using multiple transmit angles. The presence of grating lobes produces clutter in images and reduces the ability to observe tissue contrast. Techniques to reduce or eliminate the presence of grating lobes for PWI using multiple angles will result in improved image quality. Null subtraction imaging (NSI) is a nonlinear beamforming technique that has been explored for improving the lateral resolution of ultrasonic imaging. However, the apodization scheme used in NSI also eliminates or greatly reduces the presence of grating lobes. Imaging tasks using NSI were evaluated in simulations and physical experiments involving tissue-mimicking phantoms and rat tumors in vivo. Images created with NSI were compared with images created using traditional delay and sum (DAS) with Hann apodization and images created using a generalized coherence factor (GCF). NSI was observed to greatly reduce the presence of grating lobes in ultrasonic images, compared to DAS with Hann and GCF, while maintaining spatial resolution and contrast in the images. Therefore, NSI can provide a novel means of creating images using PWI with multiple steering angles on clinically available linear arrays while reducing the adverse effects associated with grating lobes.

The TOG protein Stu2 is regulated by acetylation.

Stu2 in S. cerevisiae is a member of the XMAP215/Dis1/CKAP5/ch-TOG family of MAPs and has multiple functions in controlling microtubules, including microtubule polymerization, microtubule depolymerization, linking chromosomes to the kinetochore, and assembly of γ-TuSCs at the SPB. Whereas phosphorylation has been shown to be critical for Stu2 localization at the kinetochore, other regulatory mechanisms that control Stu2 function are still poorly understood. Here, we show that a novel form of Stu2 regulation occurs through the acetylation of three lysine residues at K252, K469, and K870, which are located in three distinct domains of Stu2. Alteration of acetylation through acetyl-mimetic and acetyl-blocking mutations did not impact the essential function of Stu2. Instead, these mutations lead to a decrease in chromosome stability, as well as changes in resistance to the microtubule depolymerization drug, benomyl. In agreement with our in silico modeling, several acetylation-mimetic mutants displayed increased interactions with γ-tubulin. Taken together, these data suggest that Stu2 acetylation can govern multiple Stu2 functions, including chromosome stability and interactions at the SPB.

Identifying and overcoming limitations with in situ calibration beads for quantitative ultrasound.

Ensuring the consistency of spectral-based quantitative ultrasound estimates in vivo necessitates accounting for diffraction, system effects, and propagation losses encountered in the tissue. Accounting for diffraction and system effects is typically achieved through planar reflector or reference phantom methods; however, neither of these is able to account for the tissue losses present in vivo between the ultrasound probe and the region of interest. In previous work, the feasibility of small titanium beads as in situ calibration targets (0.5-2 mm in diameter) was investigated. In this study, the importance of bead size for the calibration signal, the role of multiple echoes coming from the calibration bead, and sampling of the bead signal laterally through beam translation were examined. This work demonstrates that although the titanium beads naturally produce multiple reverberant echoes, time-windowing of the first echo provides the smoothest calibration spectrum for backscatter coefficient calculation. When translating the beam across the bead, the amplitude of the echo decreases rapidly as the beam moves across and past the bead. Therefore, to obtain consistent calibration signals from the bead, lateral interpolation is needed to approximate signals coming from the center of the bead with respect to the beam.

A 10% Tomato Diet Selectively Reduces Radiation-Induced Damage in TRAMP Mice.

BACKGROUND: Tomatoes contain carotenoids that have the potential to alter the effects of external beam radiation therapy (EBRT). OBJECTIVES: We hypothesized that dietary lyophilized tomato paste (TP) would reduce apoptosis within carotenoid-containing nonneoplastic tissues in EBRT-treated TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice. METHODS: Male TRAMP mice (n = 73) were provided an AIN-93G diet or a modified AIN-93G diet containing 10% TP (wt:wt) at 4 wk of age. Prostate tumor growth was monitored by ultrasound. The caudal half of the mouse was irradiated with 7.5 Gy (Rad) or 0 Gy (sham) at 24 wk of age or after the tumor volume exceeded 1000 mm3 with a Cobalt-60 source. Mice were euthanized 24 h postradiation. Carotenoids and α-tocopherol were measured by HPLC and compared by a t test. Tissues were assessed for radiation-induced changes (hematoxylin and eosin) and apoptosis [cleaved caspase-3 (CC3)] and compared by Kruskal-Wallis test or Freedman-Lane's permutation test. RESULTS: Serum concentrations of lycopene (52% lower), phytoene (26% lower), and α-tocopherol (22% lower) were decreased in TP-fed irradiated mice (TP-Rad) compared with TP-fed sham mice (P < 0.05). CC3 scores increased within the prostate tumor with radiation treatments (P < 0.05), but were not affected by tomato consumption. In nonneoplastic tissues, TP-Rad had a lower percentage of CC3-positive cells within the cranial (67% lower) and caudal (75% lower) duodenum than irradiated mice on the control diet (Rad) (P < 0.005). Likewise, CC3 scores within the dorsolateral prostate of TP-Rad trended toward lower scores than for Rad (P = 0.07). CONCLUSIONS: TP selectively reduces radiation-induced apoptosis in extratumoral tissues without decreasing radiation-induced apoptosis within the prostate tumor in TRAMP mice. Additional studies are needed to confirm and expand upon these findings.

Super-Resolution Ultrasound Localization Microscopy on a Rabbit Liver VX2 Tumor Model: An Initial Feasibility Study.

Ultrasound localization microscopy can image microvasculature in vivo without sacrificing imaging penetration depth. However, the reliance on super-resolution inference limits the applicability of the technique because subpixel tissue motion can corrupt microvascular reconstruction. Consequently, the majority of previous pre-clinical research on this super-resolution procedure has been restricted to low-motion experimental models with ample motion correction or data rejection, which precludes the imaging of organ sites that exhibit a high degree of respiratory and other motion. In this article, we present a novel anesthesia protocol in rabbits that induces safe, controllable periods of apnea to enable the long image-acquisition times required for ultrasound localization microscopy. We apply this protocol to a VX2 liver tumor model undergoing sorafenib therapy and compare the results to super-resolution images from conventional high-dose isoflurane anesthesia. We find that the apneic protocol was necessary to correctly identify the poorly vascularized tumor cores, as verified by immunohistochemistry, and to reveal the tumoral microvascular architecture.

High Data Rate Communications In Vivo Using Ultrasound.

The emergence of in-body medical devices has provided a means of capturing physiological or diagnostic information and streaming this information outside of the body. Currently, electromagnetic-based communications make up the bulk of in-body medical device communication protocols. Traditional electromagnetic-based solutions are limited in their data rates and available power. Recently, ultrasound was investigated as a communication channel for through-tissue data transmission. To achieve real-time video streaming through tissue, data rates of ultrasound need to exceed 1 Mbps. In a previous study, we demonstrated ultrasound communications with data rates greater than 30 Mbps with two focused ultrasound transducers using a large footprint laboratory system through slabs of lossy tissues. While the form factor of the transmitter is also crucial, it is obvious that a large, focused transducer cannot fit within the size of a small in-body medical device. Several other challenges for achieving high-speed ultrasonic communication through tissue include strong reflections leading to multipath effects and attenuation. In this work, we demonstrate ultrasonic video communications using a mm-scale microcrystal transmitter with video streaming supplied by a camera connected to a Field Programmable Gate Array (FPGA). The signals were transmitted through a tissue-mimicking phantom and through the abdomen of a rabbit in vivo. The ultrasound signal was recorded by an array probe connected to a Verasonics Vantage system and decoded back to video. To improve the received signal quality, we combined the signal from multiple channels of the array probe. Orthogonal frequency division multiplexing (OFDM) modulation was used to reduce the receiver complexity under a strong multipath environment.

Use of a convolutional neural network and quantitative ultrasound for diagnosis of fatty liver.

Quantitative ultrasound (QUS) was used to classify rabbits that were induced to have liver disease by placing them on a fatty diet for a defined duration and/or periodically injecting them with CCl4. The ground truth of the liver state was based on lipid liver percents estimated via the Folch assay and hydroxyproline concentration to quantify fibrosis. Rabbits were scanned ultrasonically in vivo using a SonixOne scanner and an L9-4/38 linear array. Liver fat percentage was classified based on the ultrasonic backscattered radiofrequency (RF) signals from the livers using either QUS or a 1-D convolutional neural network (CNN). Use of QUS parameters with linear regression and canonical correlation analysis demonstrated that the QUS parameters could differentiate between livers with lipid levels above or below 5%. However, the QUS parameters were not sensitive to fibrosis. The CNN was implemented by analyzing raw RF ultrasound signals without using separate reference data. The CNN outputs the classification of liver as either above or below a threshold of 5% fat level in the liver. The CNN outperformed the classification utilizing the QUS parameters combined with a support vector machine in differentiating between low and high lipid liver levels (i.e., accuracies of 74% versus 59% on the testing data). Therefore, although the CNN did not provide a physical interpretation of the tissue properties (e.g., attenuation of the medium or scatterer properties) the CNN had much higher accuracy in predicting fatty liver state and did not require an external reference scan.

Video-Capable Ultrasonic Wireless Communications Through Biological Tissues.

The use of wireless implanted medical devices (IMDs) is growing because they facilitate monitoring of patients at home and during normal activities, reduce the discomfort of patients, and reduce the likelihood of infection associated with trailing wires. Currently, radio frequency (RF) electromagnetic waves are the most commonly used method for communicating wirelessly with IMDs. However, due to the restrictions on the available bandwidth and the employable power, data rates of RF-based IMDs are limited to 267 kb/s. Considering standard definition video streaming requires data rates of 1.2 Mb/s and high definition requires 3 Mb/s, it is not possible to use the RF electromagnetic communications for high data rate communication applications such as video streaming. In this work, an alternative method that utilizes ultrasonic waves to relay information at high data rates is introduced. An advanced quadrature amplitude modulation (QAM) modem with phase-compensating, sparse decision feedback equalizer (DFE) is tailored to realize the full potential of the ultrasonic channel through biological tissues. The proposed system is tested in a variety of scenarios, including both simulations with finite impulse response (FIR) channel models, and real physical transmission experiments with ex vivo beef liver and pork chop samples as well as in situ rabbit abdomen. Consequently, the simulations demonstrated that video-capable data rates can be achieved with millimeter-sized transducers. Real physical experiments confirmed data rates of 6.7, 4.4, 4, and 3.2 Mb/s through water, ex vivo beef liver, ex vivo pork chop, and in situ rabbit abdomen, respectively.

BIP and the unfolded protein response are important for potyvirus and potexvirus infection.

Plant potexvirus and potyvirus infection can trigger endoplasmic reticulum (ER) stress. ER stress signaling increases the expression of cytoprotective ER-chaperones, especially the BiP chaperones which contribute to pro-survival functions when plants are subjected to infection. The inositol requiring enzyme (IRE1) is one ER stress sensor that is activated to splice the bZIP60 mRNA which produces a truncated transcription factor that activates gene expression in the nucleus. The IRE1/bZIP60 pathway is associated with restricting potyvirus and potexvirus infection. Recent data also identified the IRE1-independent UPR pathways led by bZIP28 and bZIP17 contribute to potexvirus and potyvirus infection. These three bZIP pathways recognize cis-regulatory elements in the BiP promoters to enhance gene expression. BiP is part of a negative feedback loop that regulates the activities of the ER stress transducers IRE1, bZIP28, and bZIP17 to block their activation. We discuss a model in which bZIP60 and bZIP17 synergistically induce BiP and other genes restricting Plantago asiatica mosaic virus (PlAMV; a potexvirus) infection while bZIP60 and bZIP28 independently induce genes supporting PlAMV infection. Regarding Turnip mosiac virus (TuMV, a potyvirus) infection, bZIP60 and bZIP28 serve to repress local and systemic infection. Finally, tauroursodeoxycholic acid treatments were used to demonstrate that the protein folding capacity significantly influences PlAMV accumulation.

Genome-wide identification and characterization of Solanum tuberosum BiP genes reveal the role of the promoter architecture in BiP gene diversity.

The endoplasmic reticulum (ER) immunoglobulin binding proteins (BiPs) are molecular chaperones involved in normal protein maturation and refolding malformed proteins through the unfolded protein response (UPR). Plant BiPs belong to a multi-gene family contributing to development, immunity, and responses to environmental stresses. This study identified three BiP homologs in the Solanum tuberosum (potato) genome using phylogenetic, amino acid sequence, 3-D protein modeling, and gene structure analysis. These analyses revealed that StBiP1 and StBiP2 grouped with AtBiP2, whereas StBiP3 grouped with AtBiP3. While the protein sequences and folding structures are highly similar, these StBiPs are distinguishable by their expression patterns in different tissues and in response to environmental stressors such as treatment with heat, chemicals, or virus elicitors of UPR. Ab initio promoter analysis revealed that potato and Arabidopsis BiP1 and BiP2 promoters were highly enriched with cis-regulatory elements (CREs) linked to developmental processes, whereas BiP3 promoters were enriched with stress related CREs. The frequency and linear distribution of these CREs produced two phylogenetic branches that further resolve the groups identified through gene phylogeny and exon/intron phase analysis. These data reveal that the CRE architecture of BiP promoters potentially define their spatio-temporal expression patterns under developmental and stress related cues.

Dietary Tomato or Lycopene Do Not Reduce Castration-Resistant Prostate Cancer Progression in a Murine Model.

BACKGROUND: Dietary tomato products or lycopene protect against prostate carcinogenesis, but their impact on the emergence of castration-resistant prostate cancer (CRPC) is unknown. OBJECTIVE: We hypothesized that tomato or lycopene products would reduce the emergence of CRPC. METHODS: Transgenic adenocarcinoma of the mouse prostate (TRAMP) mice were castrated at 12-13 wk and the emergence of CRPC was monitored by ultrasound in each study. In Study 1, TRAMP mice (n = 80) were weaned onto an AIN-93G-based control diet (Con-L, n = 28), a 10% tomato powder diet (TP-L, 10% lyophilized w/w, n = 26), or a control diet followed by a tomato powder diet after castration (TP-Int1, n = 26). In Study 2, TRAMP mice (n = 85) were randomized onto a control diet with placebo beadlets (Con-Int, n = 29), a tomato diet with placebo beadlets (TP-Int2, n = 29), or a control diet with lycopene beadlets (Lyc-Int, n = 27) following castration (aged 12 wk). Tumor incidence and growth were monitored by ultrasound beginning at an age of 10 wk. Mice were euthanized 4 wk after tumor detection or aged 30 wk if no tumor was detected. Tissue weights were compared by ANOVA followed by Dunnett's test. Tumor volumes were compared using generalized linear mixed model regression. RESULTS: Ultrasound estimates for the in vivo tumor volume were strongly correlated with tumor weight at necropsy (R2 = 0.75 and 0.94, P <0.001 for both Studies 1 and 2, respectively). Dietary treatments after castration did not significantly impact cancer incidence, time to tumor detection, or final tumor weight. CONCLUSIONS: In contrast to studies of de novo carcinogenesis in multiple preclinical models, tomato components had no significant impact on the emergence of CRPC in the TRAMP model. It is possible that specific mutant subclones of prostate cancer may continue to show some antiproliferative response to tomato components, but further studies are needed to confirm this.

Depletion of hepatic forkhead box O1 does not affect cholelithiasis in male and female mice.

Cholelithiasis is one of the most prevalent gastroenterological diseases and is characterized by the formation of gallstones in the gallbladder. Both clinical and preclinical data indicate that obesity, along with comorbidity insulin resistance, is a predisposing factor for cholelithiasis. Forkhead box O1 (FoxO1) is a key transcription factor that integrates insulin signaling with hepatic metabolism and becomes deregulated in the insulin-resistant liver, contributing to dyslipidemia in obesity. To gain mechanistic insights into how insulin resistance is linked to cholelithiasis, here we determined FoxO1's role in bile acid homeostasis and its contribution to cholelithiasis. We hypothesized that hepatic FoxO1 deregulation links insulin resistance to impaired bile acid metabolism and cholelithiasis. To address this hypothesis, we used the FoxO1LoxP/LoxP-Albumin-Cre system to generate liver-specific FoxO1-knockout mice. FoxO1-knockout mice and age- and sex-matched WT littermates were fed a lithogenic diet, and bile acid metabolism and gallstone formation were assessed in these animals. We showed that FoxO1 affected bile acid homeostasis by regulating hepatic expression of key enzymes in bile acid synthesis and in biliary cholesterol and phospholipid secretion. Furthermore, FoxO1 inhibited hepatic expression of the bile acid receptor farnesoid X receptor and thereby counteracted hepatic farnesoid X receptor signaling. Nonetheless, hepatic FoxO1 depletion neither affected the onset of gallstone disease nor impacted the disease progression, as FoxO1-knockout and control mice of both sexes had similar gallstone weights and incidence rates. These results argue against the notion that FoxO1 is a link between insulin resistance and cholelithiasis.

Characterizing Fatty Liver in vivo in Rabbits, Using Quantitative Ultrasound.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease and can often lead to fibrosis, cirrhosis, cancer and complete liver failure. Liver biopsy is the current standard of care to quantify hepatic steatosis, but it comes with increased patient risk and only samples a small portion of the liver. Imaging approaches to assess NAFLD include proton density fat fraction estimated via magnetic resonance imaging (MRI) and shear wave elastography. However, MRI is expensive and shear wave elastography is not proven to be sensitive to fat content of the liver (Kramer et al. 2016). On the other hand, ultrasonic attenuation and the backscatter coefficient (BSC) have been observed to be sensitive to levels of fat in the liver (Lin et al. 2015; Paige et al. 2017). In this study, we assessed the use of attenuation and the BSC to quantify hepatic steatosis in vivo in a rabbit model of fatty liver. Rabbits were maintained on a high-fat diet for 0, 1, 2, 3 or 6 wk, with 3 rabbits per diet group (total N = 15). An array transducer (L9-4) with a center frequency of 4.5 MHz connected to a SonixOne scanner was used to gather radio frequency (RF) backscattered data in vivo from rabbits. The RF signals were used to estimate an average attenuation and BSC for each rabbit. Two approaches were used to parameterize the BSC (i.e., the effective scatterer diameter and effective acoustic concentration using a spherical Gaussian model and a model-free approach using a principal component analysis [PCA]). The 2 major components of the PCA from the BSCs, which captured 96% of the variance of the transformed data, were used to generate input features to a support vector machine for classification. Rabbits were separated into two liver fat-level classes, such that approximately half of the rabbits were in the low-lipid class (≤9% lipid liver level) and half of the rabbits in the high-lipid class (>9% lipid liver level). The slope and the midband fit of the attenuation coefficient provided statistically significant differences (p value = 0.00014 and p value = 0.007, using a two-sample t test) between low and high-lipid fat classes. The proposed model-free and model-based parameterization of the BSC and attenuation coefficient parameters yielded classification accuracies of 84.11 %, 82.93 % and 78.91 % for differentiating low-lipid versus high-lipid classes, respectively. The results suggest that attenuation and BSC analysis can differentiate low-fat versus high-fat livers in a rabbit model of fatty liver disease.

Low fat but not soy protein isolate was an effective intervention to reduce nonalcoholic fatty liver disease progression in C57BL/6J mice: monitored by a novel quantitative ultrasound (QUS) method.

Untreated nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) lead to irreversible liver damage. We hypothesized that a low-fat diet (LFD) or a high-fat diet (HFD) with soy protein isolate (SPI) would be an effective intervention to halt or reverse NAFLD progression. To test these hypotheses, we conducted 2 studies. In the first study, we fed an HFD to 7-week-old C57BL/6J mice to induce NAFLD compared to an LFD (control). Hepatic steatosis was monitored by quantitative ultrasound (QUS) scans (in vivo and ex vivo). Animals were euthanized after 0, 2, 4, and 6 weeks of feeding. In the second study, 7-week-old mice were randomized onto an LFD or HFD with SPI intervention after 4 weeks of feeding HFD. Animals from each group were scanned with QUS and euthanized after 4, 9, and 12 weeks of feeding. Animals fed the HFD developed NAFLD (100%) and NASH (80%) characterized by increased liver weight, lipid accumulation, and histological scores for inflammation by 4 weeks in the first study. In the second study, the LFD ameliorated this NAFLD phenotype after 5 weeks of feeding; however, the SPI intervention failed to significantly attenuate NAFLD. QUS parameters were significantly increased with the HFDs (P < .05) and steatosis grade (P < .05) and were positively correlated with hepatic lipid concentrations. In conclusion, dietary modification may be effective at reversing NAFLD and NASH at early stages. Furthermore, QUS may become a valuable tool to track hepatic steatosis. Additional studies are needed to further evaluate the effectiveness of these interventions.

Quantitative Ultrasound and the Pancreas: Demonstration of Early Detection Capability.

OBJECTIVES: To show that quantitative ultrasound biomarkers attenuation (AC) and backscatter (BSC) coefficients are effective tools to detect early changes in acute pancreatitis, using a cerulein-induced pancreatitis rat model. METHODS: Sprague-Dawley rats (n = 68) were divided into 8 groups: uninjected cage controls, saline-injected controls, and cerulein-injected rats euthanized at 2, 4, 15, 24, 48, and 60 hours after injection. Pancreatic AC and BSC (25-55 MHz) were estimated in vivo (Vevo 2100, VisualSonics, Toronto, CA) and ex vivo (40-MHz transducer). The pancreas of each rat was evaluated histopathologically. RESULTS: Changes in both in vivo and ex vivo AC and BSC relative to controls reflected temporal histomorphologic changes. Overall, there were decreased AC and BSC at early time points and then rebound toward control values over time. Maximal in vivo AC and BSC decreases occurred at 2 hours after cerulein injection. Attenuation coefficient changes corresponded well with early pancreatic edema and acinar cell vacuolation, with rebound as edema decreased, autophagy/cellular death occurred, and histiocytic infiltrates and fibrosis manifested. Backscatter coefficient decreased early but rebounded as autophagy and apoptosis increased, only to fall as acinar atrophy peaked, and fibrosis and histiocytic infiltration increased. CONCLUSIONS: Cerulein-induced pancreatitis is an excellent model for studying ultrasonic AC and BSC biomarkers during the early stages of acute pancreatitits, reflecting microscopic structural changes. Edema followed by cell shrinkage and apoptosis, then histiocytic infiltration and fibrosis, has certain similarities with the morphologies of some forms of pancreatic carcinoma. This suggests that quantitative ultrasound may be very useful for early detection of disease onset or response to therapy for not only acute pancreatitis but also pancreatic cancer.