Comparing Focused-Tracked and Plane Wave-Tracked ARFI Log(VoA) In Silico and in Application to Human Carotid Atherosclerotic Plaque, Ex Vivo.

A significant risk factor for ischemic stroke is carotid atherosclerotic plaque that is susceptible to rupture, with rupture potential conveyed by plaque morphology. Human carotid plaque composition and structure have been delineated noninvasively and in vivo by evaluating log(VoA), a parameter derived as the decadic log of the second time derivative of displacement induced by an acoustic radiation force impulse (ARFI). In prior work, ARFI-induced displacement was measured using conventional focused tracking; however, this requires a long data acquisition period, thereby reducing framerate. We herein evaluate if ARFI log(VoA) framerate can be increased without a reduction in plaque imaging performance using plane wave tracking instead. In silico, both focused- and plane wave-tracked log(VoA) decreased with increasing echobrightness, quantified as signal-to-noise ratio (SNR), but did not vary with material elasticity for SNRs below 40 dB. For SNRs of 40-60 dB, both focused- and plane wave-tracked log(VoA) varied with SNR and material elasticity. Above 60 dB SNR, both focused- and plane wave-tracked log(VoA) varied with material elasticity alone. This suggests that log(VoA) discriminates features according to a combination of their echobrightness and mechanical property. Further, while both focused- and plane-wave tracked log(VoA) values were artifactually inflated by mechanical reflections at inclusion boundaries, plane wave-tracked log(VoA) was more strongly impacted by off-axis scattering. Applied to three excised human cadaveric carotid plaques with spatially aligned histological validation, both log(VoA) methods detected regions of lipid, collagen, and calcium (CAL) deposits. These findings support that plane wave tracking performs comparably to focused tracking for log(VoA) imaging and that plane wave-tracked log(VoA) is a viable approach to discriminating clinically relevant atherosclerotic plaque features at a 30-fold higher framerate than by focused tracking.

Zinc Binding Inhibits Cellular Uptake and Antifungal Activity of Histatin-5 in Candida albicans.

Histatin-5 (Hist-5) is a polycationic, histidine-rich antimicrobial peptide with potent antifungal activity against the opportunistic fungal pathogen Candida albicans. Hist-5 can bind metals in vitro, and metals have been shown to alter the fungicidal activity of the peptide. Previous reports on the effect of Zn2+ on Hist-5 activity have been varied and seemingly contradictory. Here, we present data elucidating the dynamic role Zn2+ plays as an inhibitory switch to regulate Hist-5 fungicidal activity. A novel fluorescently labeled Hist-5 peptide (Hist-5*) was developed to visualize changes in internalization and localization of the peptide as a function of metal availability in the growth medium. Hist-5* was verified for use as a model peptide and retained antifungal activity and mode of action similar to native Hist-5. Cellular growth assays showed that Zn2+ had a concentration-dependent inhibitory effect on Hist-5 antifungal activity. Imaging by confocal microscopy revealed that equimolar concentrations of Zn2+ kept the peptide localized along the cell periphery rather than internalizing, thus preventing cytotoxicity and membrane disruption. However, the Zn-induced decrease in Hist-5 activity and uptake was rescued by decreasing the Zn2+ availability upon addition of a metal chelator EDTA or S100A12, a Zn-binding protein involved in the innate immune response. These results lead us to suggest a model wherein commensal C. albicans may exist in harmony with Hist-5 at concentrations of Zn2+ that inhibit peptide internalization and antifungal activity. Activation of host immune processes that initiate Zn-sequestering mechanisms of nutritional immunity could trigger Hist-5 internalization and cell killing.

Development of a Robotic Shear Wave Elastography System for Noninvasive Staging of Liver Disease in Murine Models.

Shear wave elastography (SWE) is an ultrasound-based stiffness quantification technology that is used for noninvasive liver fibrosis assessment. However, despite widescale clinical adoption, SWE is largely unused by preclinical researchers and drug developers for studies of liver disease progression in small animal models due to significant experimental, technical, and reproducibility challenges. Therefore, the aim of this work was to develop a tool designed specifically for assessing liver stiffness and echogenicity in small animals to better enable longitudinal preclinical studies. A high-frequency linear array transducer (12-24 MHz) was integrated into a robotic small animal ultrasound system (Vega; SonoVol, Inc., Durham, NC) to perform liver stiffness and echogenicity measurements in three dimensions. The instrument was validated with tissue-mimicking phantoms and a mouse model of nonalcoholic steatohepatitis. Female C57BL/6J mice (n = 40) were placed on choline-deficient, L-amino acid-defined, high-fat diet and imaged longitudinally for 15 weeks. A subset was sacrificed after each imaging timepoint (n = 5) for histological validation, and analyses of receiver operating characteristic (ROC) curves were performed. Results demonstrated that robotic measurements of echogenicity and stiffness were most strongly correlated with macrovesicular steatosis (R2  = 0.891) and fibrosis (R2  = 0.839), respectively. For diagnostic classification of fibrosis (Ishak score), areas under ROC (AUROCs) curves were 0.969 for ≥Ishak1, 0.984 for ≥Ishak2, 0.980 for ≥Ishak3, and 0.969 for ≥Ishak4. For classification of macrovesicular steatosis (S-score), AUROCs were 1.00 for ≥S2 and 0.997 for ≥S3. Average scanning and analysis time was <5 minutes/liver. Conclusion: Robotic SWE in small animals is feasible and sensitive to small changes in liver disease state, facilitating in vivo staging of rodent liver disease with minimal sonographic expertise.