Search

Although sonographic artifacts are common in sonography, they can create diagnostic challenges and even lead to critical diagnostic errors. The authors report a case of an echogenic artifact resembling an abdominal aortic thrombus. An awareness of the existence of this pseudothrombus and techniques to differentiate this artifact from an actual fibrinous clot may help avoid unnecessary anxiety and costly testing. [ABSTRACT FROM AUTHOR]

The idea that much of nursing is 'ritualized' activity which is harmful to patient care assumes that 'ritual' itself is unacceptable behaviour or practice. At a time when market forces are clearly influencing the delivery of care and, in turn, changes in nursing practice, it has become important both to clarify what 'ritual' is and to determine its existence and 'form' within nursing. This study explored nursing culture for 'ritual' in a ward setting and used ethnography as both method and description. Rituals were found to exist in the working day of the nurses studied, but was not an indication that 'ritualized behaviour' is harmful to individualized patient care. There is a clear need, however, to determine specifically the difference between 'unsafe outdated practices' and ritual in a cultural 'sense'. This would ensure that what had to be relinquished would in no way jeopardize the future existence of nursing and nurses as socially cohesive groups with their own culture. [ABSTRACT FROM AUTHOR]

Background: The call for quality improvement and patient safety (QI/PS) education has increased at every level of medical education. Here, the authors present a QI/PS experiential platform implemented at the University of Florida College of Medicine (UFCOM). The project established a peer-taught hands-on learning platform in a student-run clinic allowing participants to learn and apply QI/PS concepts and tools in a real-world clinic environment. The aims were to assess students' perceptions in regard to (1) student confidence in quality improvement (QI) methodology, and (2) competency in executing QI initiatives in healthcare as measured by a post-participation survey., Method: A medical student-led quality improvement team was embedded within University of Florida's (UF's) existing student-run clinic network. The QI/PS student-team collaborated with clinic leaders and utilized QI/PS tools to establish, monitor, and expand impactful Plan-Do-Study-Act (PDSA) cycles. The impact of the training was then evaluated using The New World Kirkpatrick Model leveraging a post-project survey which included the Beliefs, Attitudes, Skills, and Confidence in Quality Improvement (BASiC-QI) survey and questions on overall student perceptions., Results: This project demonstrated positive results in all four levels of Kirkpatrick evaluation: (1) Reaction, (2) Learning, (3) Behavior, and (4) Results. This was shown through (1) a voluntary feedback survey that reported positive feedback from participants with 93% of respondents indicating they "strongly agreed" or "agreed" to positive perception questions; (2) significantly higher scores (p < 0.001) on the BASiC-QI Scale for project participants vs. non-participants; (3) the completion of 4.25 PDSA cycles per QI team; and (4) a 10.1% reduction in median patient time in clinic., Conclusions: This study supports the utility of incorporating a student-led QI/PS interactive platform into student-run clinics to increase knowledge and attitude in implementing QI/PS endeavors while simultaneously improving clinic metrics and outcomes for patients., Competing Interests: Declarations. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Objective: Acoustic droplet vaporization (ADV) is the liquid-to-gas phase transition of perfluorocarbon (PFC) droplets to microbubbles upon ultrasound insonation. After ADV, gases dissolved in the surrounding fluid diffuse into microbubbles, enabling oxygen scavenging. Characterization of oxygen scavenging and transition efficiency (TE) in whole blood has so far been limited. In this work, oxygen scavenging and perfluorocarbon droplet TE in a saline buffer and whole bovine blood were evaluated using blood-gas analysis and flow cytometry., Methods: Oxygen scavenging from whole blood via ADV was determined using an in vitro flow phantom with droplets comprising a phospholipid shell and either a decafluorobutane (DFB) or a perfluoropentane (PFP) core. Fluorescent droplets were used to determine ADV TE in whole blood via flow cytometry. Finally, a mathematical model predicting oxygen scavenging from whole blood was developed based on the experimental TE values., Results: DFB droplets enabled greater oxygen scavenging and higher TE when compared with perfluoropentane droplets in both buffer and whole blood. Increasing the droplet concentration resulted in a greater amount of hemoglobin-bound and dissolved oxygen scavenging from whole blood. ADV of DFB droplets at a concentration of 5 × 10 -4 mL/mL yielded a total oxygen reduction of 913 μM. The TE decreased with increasing droplet concentration in both buffer and whole blood. Experimental oxygen scavenging data in whole blood aligned with the predicted values from the mathematical model., Conclusion: Increased oxygen scavenging and TE were achieved with DFB droplets relative to perfluoropentane droplets., Competing Interests: Conflict of Interest C.K.H. and K.J.H. have been consultants with Boston Scientific Inc. All other authors have no conflicts of interest to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: The hypothesis of this study was histotripsy, an ultrasound therapy that disrupts tissue mechanically through the action of bubble clouds, increases the short-term rate of acute thrombus clearance for catheter-directed thrombolysis (CDT) in an animal model., Materials and Methods: Thrombi formed in the femoral vein of pigs were treated with CDT, histotripsy, or CDT and histotripsy (histotripsy+). Ultrasound (B-mode and color Doppler) and contrast fluoroscopy imaging data were scored by four observers for semi-quantitative evaluation of each arm with ordinal regression models. Further, B-mode images were manually annotated by three observers to quantify the thrombus clearance rate., Results: A total of 27 thrombi (2.0 ± 0.4 cm in length) in 27 animals were considered in this study (N = 8 for CDT, N = 9 for histotripsy, and N = 10 for histotripsy+). The mean treatment duration was 20.2 ± 1.3 min. The ordinal regression models indicated the thrombus clearance rate was increased for histotripsy+ relative to CDT based on B-mode and color Doppler, but not fluoroscopy (p = 0.015, 0.001, and 0.900, respectively). Manual annotation of B-mode images denoted histotripsy+ had an increased thrombus clearance rate relative to CDT and histotripsy (p = 0.001 and 0.022, respectively). Petechial hemorrhage was present in the perivascular soft tissue for two cases with histotripsy and one case with histotripsy+., Conclusions: The clearance of acute thrombus was similar for treatment with CDT or histotripsy. Combining these individual approaches further increased the rate of thrombus clearance based on multiple imaging metrics., (Copyright © 2025. Published by Elsevier Inc.)

Background: The University of Florida (UF) Equal Access Clinic Network (EACN) is the largest student-run free healthcare clinic network in Florida. The UF EACN serves those who are underinsured or uninsured in Alachua County and its surrounding area. Nationally, average total clinic time per medical visit has been established to be 84 min., Problem: Before this project, average patient cycle time at the UF EACN was 125.3 min, and there was no established quality improvement (QI) team to implement changes to address inefficiencies., Methods: This was a prospective QI study that recorded patient cycle times for patients who received healthcare at any of the four primary care free clinics across the UF EACN from 5 July 2022 to 6 April 2023., Interventions: Eighteen Plan-Do-Study-Act cycles were tailored to each of the four primary care clinic's needs with a focus on reducing patient cycle time by addressing the following identified problems: prolonged intake process, translation services, limited numbers of volunteers, and other inefficiencies and bottlenecks in workflow., Results: The median patient cycle time at the EACN shifted from 125.3 min to 112.7 min over a nine month period. This drop of 12.6 min meant patients saw a 10.1% reduction in patient cycle time across the EACN., Conclusion: Underserved patients at EACN are experiencing increased value by having shorter patient cycle times., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Plants synthesize an array of volatile compounds, many of which serve ecological roles in attracting pollinators, deterring herbivores, and communicating with their surroundings. Methyl anthranilate (MeAA) is an anti-herbivory defensive volatile responsible for grape aroma that is emitted by several agriculturally relevant plants, including citrus, grapes, and maize. Unlike maize, which uses a one-step anthranilate methyltransferase (AAMT), grapes have been thought to use a two-step pathway for MeAA biosynthesis. By mining available transcriptomics data, we identified two AAMTs in Vitis vinifera (wine grape), as well as one ortholog in "Concord" grape. Many angiosperms methylate the plant hormone salicylic acid (SA) to produce methyl salicylate, which acts as a plant-to-plant communication molecule. Because the Citrus sinensis (sweet orange) SA methyltransferase can methylate both anthranilate (AA) and SA, we used this enzyme to examine the molecular basis of AA activity by introducing rational mutations, which identified several active site residues that increase activity with AA. Reversing this approach, we introduced mutations that imparted activity with SA in the maize AAMT, which uncovered different active site residues from those in the citrus enzyme. Sequence and phylogenetic analysis revealed that one of the Vitis AAMTs shares an ancestor with jasmonic acid methyltransferases, similar to the AAMT from strawberry (Frageria sp.). Collectively, these data demonstrate the molecular mechanisms underpinning AA activity across methyltransferases and identify one-step enzymes by which grapes synthesize MeAA., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Objectives: In competitive motocross, children as young as 4 years old race in groups on motorized off-road bikes on uneven terrain. We aimed to describe pediatric injuries occurring during an annual week-long certified amateur motocross competition between 2011 and 2021. Secondarily, we compared injury characteristics and medical evaluation by age., Methods: This retrospective analysis of injuries sustained by children during an annual motocross competition included children <18 years who received care for an event-related injury within either of the two large regional hospital systems between 2011 and 2021. Data were collected through electronic health record review and analyzed with descriptive statistics. We used chi-square and Fisher exact tests to compare findings by age (young child less than 12 years vs. adolescent 12 years or older)., Results: Over the 10-week study period (1 week per year for each of 10 years), 286 encounters were made by 278 children. Nearly all children (280/286, 98%) underwent imaging; most had at least one traumatic finding (71.7% of x-rays, 62.4% of computed tomography [CT] scans). Ninety-three children (32.5% of 286) sustained multisystem injuries. Emergency department procedures included one endotracheal intubation, one thoracostomy, 46 closed reductions, and 37 procedural sedations. Twenty-eight children (9.8% of 286) required operative intervention. Overall, 25.5% of children (73/286) were hospitalized and one adolescent died. Adolescents were more likely than young children to undergo CT imaging (40.1% vs. 26.8%, p  = 0.042) and have multisystem injuries (36.3% vs. 23.2%, p  = 0.045). There was no difference in hospitalization or operative intervention by age., Conclusion: This comprehensive assessment of injuries sustained by children during competitive motocross demonstrates significant morbidity and mortality. Findings have implications for families who consider participation and health systems in regions where competitions occur., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s). Journal of the American College of Emergency Physicians Open published by Wiley Periodicals LLC on behalf of American College of Emergency Physicians.)

Objective: Standard treatment for deep vein thrombosis (DVT) involves catheter-directed anticoagulants or thrombolytics, but the chronic thrombi present in many DVT cases are often resistant to this therapy. Histotripsy has been found to be a promising adjuvant treatment, using the mechanical action of cavitating bubble clouds to enhance thrombolytic activity. The objective of this study was to determine if histotripsy enhanced recombinant tissue plasminogen activator (rt-PA) thrombolysis in highly retracted porcine clots in vitro in a flow model of occlusive DVT., Methods: Highly retracted porcine whole blood clots were treated for 1 h with either catheter-directed saline (negative control), rt-PA (lytic control), histotripsy, DEFINITY and histotripsy or the combination of rt-PA and histotripsy with or without DEFINITY. Five-cycle, 1.5 MHz histotripsy pulses with a peak negative pressure of 33.2 MPa and pulse repetition frequency of 40 Hz were applied along the clot. B-Mode and passive cavitation images were acquired during histotripsy insonation to monitor bubble activity., Results: Clots subjected to histotripsy with and without rt-PA exhibited greater thrombolytic efficacy than controls (7.0% flow recovery or lower), and histotripsy with rt-PA was more efficacious than histotripsy with saline (86.1 ± 10.2% compared with 61.7 ± 19.8% flow recovery). The addition of DEFINITY to histotripsy with or without rt-PA did not enhance either thrombolytic efficacy or cavitation dose. Cavitation dose generally did not correlate with thrombolytic efficacy., Conclusion: Enhancement of thrombolytic efficacy was achieved using histotripsy, with and without catheter-directed rt-PA, in the presence of physiologic flow. This suggests these treatments may be effective as therapy for DVT., Competing Interests: Conflict of interest The authors have no conflicts of interest to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

GRETCHEN HAGEN 3 (GH3) acyl acid amido synthetases conjugate amino acids to acyl acid hormones to either activate or inactivate the hormone molecule. The largest subgroup of GH3 proteins modify the growth-promoting hormone auxin (indole-3-acetic acid; IAA) with the second largest class activating the defense hormone jasmonic acid (JA). The two-step reaction mechanism of GH3 proteins provides a potential proofreading mechanism to ensure fidelity of hormone modification. Examining pyrophosphate release in the first-half reaction of Arabidopsis GH3 proteins that modify IAA (AtGH3.2/YDK2, AtGH3.5/WES1, AtGH3.17/VAS2), JA (AtGH3.11/JAR1), and other acyl acids (AtGH3.7, AtGH3.12/PBS3) indicates that acyl acid-AMP intermediates are hydrolyzed into acyl acid and AMP in the absence of the amino acid, a typical feature of pre-transfer editing mechanisms. Single-turnover kinetic analysis of AtGH3.2/YDK2 and AtGH3.5/WES1 shows that non-cognate acyl acid-adenylate intermediates are more rapidly hydrolyzed than the cognate IAA-adenylate. In contrast, AtGH3.11/JAR1 only adenylates JA, not IAA. While some of the auxin-conjugating GH3 proteins in Arabidopsis (i.e., AtGH3.5/WES1) accept multiple acyl acid substrates, others, like AtGH3.2/YDK2, are specific for IAA; however, both these proteins share similar active site residues. Biochemical analysis of chimeric variants of AtGH3.2/YDK2 and AtGH3.5/WES1 indicates that the C-terminal domain contributes to selection of cognate acyl acid substrates. These findings suggest that the hydrolysis of non-cognate acyl acid-adenylate intermediates, or proofreading, proceeds via a slowed structural switch that provides a checkpoint for fidelity before the full reaction proceeds., Competing Interests: Conflict of interest J. M. J. is an associate editor of this journal. The other author declares that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Cavitation-enhanced delivery of therapeutic agents is under development for the treatment of cancer and neurodegenerative and cardiovascular diseases, including sonothrombolysis for deep vein thrombosis. The objective of this study was to quantify the spatial and temporal distribution of cavitation activity nucleated by Definity infused through the EKOS catheter over a range of acoustic parameters controlled by the EKOS endovascular system., Methods: Three insonation protocols were compared in an in vitro phantom mimicking venous flow to measure the effect of peak rarefactional pressure, pulse duration and pulse repetition frequency on cavitation activity energy, location and duration. Inertial and stable cavitation activity was quantified using passive cavitation imaging, and a metric of cavitation dose based on energy density was defined., Results: For all three insonation protocols, cavitation was sustained for the entire 30 min Definity infusion. The evolution of cavitation energy during each pulse duration was similar for all three protocols. For insonation protocols with higher peak rarefactional acoustic pressures, inertial and stable cavitation doses also increased. A complex relationship between the temporal behavior of cavitation energy within each pulse and the pulse repetition frequency affected the cavitation dose for the three insonation protocols. The relative predominance of stable or inertial cavitation dose varied according to insonation schemes. Passive cavitation images revealed the spatial distribution of cavitation activity., Conclusion: Our cavitation dose metric based on energy density enabled the impact of different acoustic parameters on cavitation activity to be measured. Depending on the type of cavitation to be promoted or suppressed, particular pulsing schemes could be employed in future studies, for example, to correlate cavitation dose with sonothrombolytic efficacy., Competing Interests: Conflict of interest Boston Scientific provided the EKOS Endovascular System control unit and catheters. C.G. is a former employee and shareholder of Boston Scientific, Inc., and C.K.H. and K.J.H. are consultants with Boston Scientific, Inc. All other authors have no conflicts of interest to disclose., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

While mammals require the essential amino acid tryptophan (Trp) in their diet, plants and microorganisms synthesize Trp de novo. The five-step Trp pathway starts with the shikimate pathway product, chorismate. Chorismate is converted to the aromatic compound anthranilate, which is then conjugated to a phosphoribosyl sugar in the second step by anthranilate phosphoribosyltransferase (PAT1). As a single-copy gene in plants, all fixed carbon flux to indole and Trp for protein synthesis, specialized metabolism, and auxin hormone biosynthesis proceeds through PAT1. While bacterial PAT1s have been studied extensively, plant PAT1s have escaped biochemical characterization. Using a structure model, we identified putative active site residues that were variable across plants and kinetically characterized six PAT1s (Arabidopsis thaliana (thale cress), Citrus sinensis (sweet orange), Pistacia vera (pistachio), Juglans regia (English walnut), Selaginella moellendorffii (spike moss), and Physcomitrium patens (spreading earth-moss)). We probed the catalytic efficiency, substrate promiscuity, and regulation of these six enzymes and found that the C. sinensis PAT1 is highly specific for its cognate substrate, anthranilate. Investigations of site-directed mutants of the A. thaliana PAT1 uncovered an active site residue that contributes to promiscuity. While Trp inhibits bacterial PAT1 enzymes, the six plant PAT1s that we tested were not modulated by Trp. Instead, the P. patens PAT1 was inhibited by tyrosine, and the S. moellendorffii PAT1 was inhibited by phenylalanine. This structure-informed biochemical examination identified variations in activity, efficiency, specificity, and enzyme-level regulation across PAT1s from evolutionarily diverse plants., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Passive cavitation imaging (PCI) with a clinical diagnostic array results in poor axial localization of bubble activity due to the size of the point spread function (PSF). The objective of this study was to determine if data-adaptive spatial filtering improved PCI beamforming performance relative to standard frequency-domain delay, sum, and integrate (DSI) or robust Capon beamforming (RCB). The overall goal was to improve source localization and image quality without sacrificing computation time. Spatial filtering was achieved by applying a pixel-based mask to DSI- or RCB-beamformed images. The masks were derived from DSI, RCB, or phase or amplitude coherence factors (ACFs) using both receiver operating characteristic (ROC) and precision-recall (PR) curve analyses. Spatially filtered passive cavitation images were formed from cavitation emissions based on two simulated sources densities and four source distribution patterns mimicking cavitation emissions induced by an EkoSonic catheter. Beamforming performance was assessed via binary classifier metrics. The difference in sensitivity, specificity, and area under the ROC curve (AUROC) differed by no more than 11% across all algorithms for both source densities and all source patterns. The computational time required for each of the three spatially filtered DSIs was two orders of magnitude less than that required for time-domain RCB and thus this data-adaptive spatial filtering strategy for PCI beamforming is preferable given the similar binary classification performance.

Ultrasound-enhanced delivery of therapeutic-loaded echogenic liposomes is under development for vascular applications using the EkoSonic Endovascular System. In this study, fibrin-targeted echogenic liposomes loaded with an anti-inflammatory agent were characterized before and after infusion through an EkoSonic catheter. Cavitation activity was nucleated by Definity or fibrin-targeted, drug-loaded echogenic liposomes infused and insonified with EkoSonic catheters. Passive cavitation imaging was used to quantify and map bubble activity in a flow phantom mimicking porcine arterial flow. Cavitation was sustained during 3-min infusions of Definity or echogenic liposomes along the distal 6 cm treatment zone of the catheter. Though the EkoSonic catheter was not designed specifically for cavitation nucleation, infusion of drug-loaded echogenic liposomes can be employed to trigger and sustain bubble activity for enhanced intravascular drug delivery., (© 2023. The Author(s).)

Surgical intervention for the treatment of intracerebral hemorrhage (ICH) has been limited by inadequate lysis of the target thrombus. Adjuvant transcranial ultrasound exposure is hypothesized to improve thrombolysis, expedite hematoma evacuation and improve clinical outcomes. A juvenile porcine intracerebral hemorrhage model was established by direct infusion of autologous blood into the porcine white matter. Thrombi were either not treated (sham) or treated with recombinant tissue plasminogen activator alone (rt-PA only) or in combination with pulsed transcranial 120-kHz ultrasound (sonothrombolysis). After treatment, pigs were euthanized, the heads frozen and sectioned and the thrombi extracted. D-Dimer and thrombus density assays were used to assess degree of lysis. Both porcine and human D-dimer assays tested did not have sufficient sensitivity to detect porcine D-dimer. Thrombi treated with rt-PA with or without 120-kHz ultrasound had a significantly lower density compared with sham-treated thrombi. No enhancement of rt-PA-mediated thrombolysis was noted with the addition of 120-kHz ultrasound (sonothrombolysis). The thrombus density assay revealed thrombolytic efficacy caused by rt-PA in an in vivo juvenile porcine model of intracerebral hemorrhage. Transcranial sonothrombolysis did not enhance rt-PA-induced thrombolysis, likely because of the lack of exogenous cavitation nuclei., Competing Interests: Conflict of interest disclosure The authors have no conflicts of interest to declare with respect to the current article., (Copyright © 2022. Published by Elsevier Inc.)

Peri-stent restenosis following stent implantation is a major clinical problem. We have previously demonstrated that ultrasound-facilitated liposomal delivery of pioglitazone (PGN) to the arterial wall attenuated in-stent restenosis. To evaluate ultrasound mediated arterial delivery, in Yucatan miniswine, balloon inflations were performed in the carotid and subclavian arteries to simulate stent implantation and induce fibrin formation. The fibrin-binding peptide, GPRPPGGGC, was conjugated to echogenic liposomes (ELIP) containing dinitrophenyl-L-alanine-labelled pioglitazone (DNP-PGN) for targeting purposes. After pre-treating the arteries with nitroglycerine, fibrin-binding peptide-conjugated PGN-loaded ELIP (PAFb-DNP-PGN-ELIP also termed atheroglitatide) were delivered to the injured arteries via an endovascular catheter with an ultrasound core, either with or without ultrasound application (EKOS TM Endovascular System, Boston Scientific). In arteries treated with atheroglitatide, there was substantial delivery of PGN into the superficial layers (5 µm from the lumen) of the arteries with and without ultrasound, [(1951.17 relative fluorescence units (RFU) vs. 1901.17 RFU; P -value = 0.939)]. With ultrasound activation there was increased penetration of PGN into the deeper arterial layers (up to 35 µm from the lumen) [(13195.25 RFU vs. 7681.00 RFU; P -value = 0.005)]. These pre-clinical data demonstrate ultrasound mediated therapeutic vascular delivery to deeper layers of the injured arterial wall. This model has the potential to reduce peri- stent restenosis.

Deep vein thrombosis is a major source of morbidity and mortality worldwide. Catheter-directed thrombolytics are the frontline approach for vessel recanalization, though fibrinolytic efficacy is limited for stiff, chronic thrombi. Although thrombin has been used in preclinical models to induce thrombosis, the effect on lytic susceptibility and clot stiffness is unknown. The goal of this study was to explore the effect of bovine thrombin concentration and incubation time on lytic susceptibility and stiffness of porcine whole blood clots in vitro. Porcine whole blood was allowed to coagulate at 37°C in glass pipets primed with 2.5 or 15 U/mL thrombin for 15 to 120 min. Lytic susceptibility to recombinant tissue plasminogen activator (rt-PA, alteplase) over a range of concentrations (3.15-107.00 µg/mL) was evaluated using percentage clot mass loss. The Young's moduli and degrees of retraction of the clots were estimated using ultrasound-based single-track-location shear wave elasticity and B-mode imaging, respectively. Percentage mass loss decreased and clot stiffness increased with the incubation period. Clots formed with 15 U/mL and incubated for 2 h exhibited properties similar to those of highly retracted clots: Young's modulus of 2.39 ± 0.36 kPa and percentage mass loss of 8.69 ± 2.72% when exposed to 3.15 µg/mL rt-PA. The histological differences between thrombin-induced porcine whole blood clots in vitro and thrombi in vivo are described., (Copyright © 2022 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

l-Tyrosine is an essential amino acid for protein synthesis and is also used in plants to synthesize diverse natural products. Plants primarily synthesize tyrosine via TyrA arogenate dehydrogenase (TyrA a or ADH), which are typically strongly feedback inhibited by tyrosine. However, two plant lineages, Fabaceae (legumes) and Caryophyllales, have TyrA enzymes that exhibit relaxed sensitivity to tyrosine inhibition and are associated with elevated production of tyrosine-derived compounds, such as betalain pigments uniquely produced in core Caryophyllales. Although we previously showed that a single D222N substitution is primarily responsible for the deregulation of legume TyrAs, it is unknown when and how the deregulated Caryophyllales TyrA emerged. Here, through phylogeny-guided TyrA structure-function analysis, we found that functionally deregulated TyrAs evolved early in the core Caryophyllales before the origin of betalains, where the E208D amino acid substitution in the active site, which is at a different and opposite location from D222N found in legume TyrAs, played a key role in the TyrA functionalization. Unlike legumes, however, additional substitutions on non-active site residues further contributed to the deregulation of TyrAs in Caryophyllales. The introduction of a mutation analogous to E208D partially deregulated tyrosine-sensitive TyrAs, such as Arabidopsis TyrA2 (AtTyrA2). Moreover, the combined introduction of D222N and E208D additively deregulated AtTyrA2, for which the expression in Nicotiana benthamiana led to highly elevated accumulation of tyrosine in planta. The present study demonstrates that phylogeny-guided characterization of key residues underlying primary metabolic innovations can provide powerful tools to boost the production of essential plant natural products., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Deep vein thrombosis is a major source of morbidity and mortality worldwide. For acute proximal deep vein thrombosis, catheter-directed thrombolytic therapy is an accepted method for vessel recanalization. Thrombolytic therapy is not without risk, including the potential for hemorrhagic bleeding that increases with lytic dose. Histotripsy is a focused ultrasound therapy that generates bubble clouds spontaneously in tissue at depth. The mechanical activity of histotripsy increases the efficacy of thrombolytic therapy at doses consistent with current pharmacomechanical treatments for venous thrombosis. The objective of this study was to determine the influence of lytic dose on histotripsy-enhanced fibrinolysis. Human whole blood clots formed in vitro were exposed to histotripsy and a thrombolytic agent (recombinant tissue plasminogen activator, rt-PA) in a venous flow model perfused with plasma. Lytic was administered into the clot via an infusion catheter at concentrations ranging from 0 (control) to 4.54 μg/mL (a common clinical dose for catheter-directed thrombolysis). Following treatment, perfusate samples were assayed for markers of fibrinolysis, hemolysis, and intact red blood cells and platelets. Fibrinolysis was equivalent between the common clinical dose of rt-PA (4.54 μg/mL) and rt-PA at a reduction to one-twentieth of the common clinical dose (0.23 μg/mL) when combined with histotripsy. Minimal changes were observed in hemolysis for treatment arms with or without histotripsy, potentially due to clot damage from insertion of the infusion catheter. Likewise, histotripsy did not increase the concentration of red blood cells or platelets in the perfusate following treatment compared to rt-PA alone. At the highest lytic dose, a refined histotripsy exposure scheme was implemented to cover larger areas of the clot. The updated exposure scheme improved clot mass loss and fibrinolysis relative to administration of lytic alone. Overall, the data collected in this study indicate the rt-PA dose can be reduced by more than a factor of ten and still promote fibrinolysis when combined with histotripsy., Competing Interests: O. A. has acted as a consultant for Inari Medical, Boston Scientific, and received research grants from Inari Medical, Canon Medical, and Philips. He acted as a speaker and received compensation for Argon Medical, Canon Medical, Penumbra, Philips, and Johnson and Johnson. G.D.W. received honoraria and serves on the advisory board for Diagnostica Stago. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Structured quality improvement and patient safety (QI/PS) education has increased at every level of medical education; however, great variability exists in the content taught. Here, the authors present a longitudinal model for medical student QI/PS education that is currently implemented at the University of Florida College of Medicine. The curriculum is taught with a variety of teaching methods incorporated into each year with increasing levels of clinical implementation. This curriculum is multimodal and introduces students to QI/PS concepts, presents mock scenarios, and eventually encourages clinical application to situations students experience during their own clinical practice. Additionally, a specialized track for students to have further immersion into this field of medicine is described, which involves specialized training, expanded educational opportunities, and a capstone project. Both the curriculum and specialized track contain explicit clinical integration to ensure students are prepared to enter the medical profession to engage in QI/PS endeavors., (Copyright © 2022 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.)

Chronic thrombi of the deep veins of the leg are resistant to dissolution or removal by current interventions and can act as thrombogenic sources. Histotripsy, a focused ultrasound therapy, uses the mechanical activity of bubble clouds to liquefy target tissues. In vitro experiments have shown that histotripsy enhances thrombolytic agent recombinant tissue plasminogen activator in a highly retracted clot model resistant to lytic therapy alone. Although these results are promising, further refinement of the acoustic source is necessary for in vivo studies and clinical translation. The source parameters for use in vivo were defined, and a transducer was fabricated for transcutaneous exposure of porcine and human iliofemoral deep-vein thrombosis (DVT) as the target. Based on the design criteria, a 1.5-MHz elliptical source with a 6-cm focal length and a focal gain of 60 was selected. The source was characterized by fiber-optic hydrophone and holography. High-speed photography showed that the cavitation cloud could be confined to dimensions smaller than the specified vessel lumen. The source was also demonstrated in vitro to create confined lesions within clots. The results support that this design offers an appropriate clinical prototype for combined histotripsy-thrombolytic therapy.

Plants are molecular factories that have spent millions of years evolving the enzymes needed to synthesize diverse primary and specialized metabolites. Despite the wealth of metabolites that plants produce, many of the enzymes responsible for generating these molecules have yet to be identified. For enzymes with known substrates, the extent of substrate promiscuity and small-molecule regulation remains unexplored. Many computational methods for identifying metabolic enzymes focus on gene-based approaches that rely on transcriptomics, metabolomics, and comparative genomics. With new AI-based tools for accurate protein structure prediction, protein-based strategies that screen a library of small molecules against a high-quality protein model can facilitate the identification of substrates, products, or inhibitors. Virtual screening has been used for structure-based drug design in the pharmaceutical industry for decades and easily translates to investigating plant metabolic enzymes. Here, we present a method for rapid, user-friendly, and open-source virtual screening using the Arabidopsis thaliana UGT74F2 with a curated library of specialized metabolites and herbicides and AutoDock Vina as an example. This method may be applied broadly to metabolic enzymes, and compound libraries can be easily adapted. Compounds are ranked based on their relative binding affinities and the resulting binding modes are evaluated using a molecular visualization program, like PyMOL. Because this is a computational approach, results from the virtual screen will need to be validated using in vitro or in vivo activity, binding, or inhibition assays. Virtual screening may aid in identifying substrates for enzymes of unknown function, revisiting substrate selectivity, or identifying natural or synthetic inhibitors., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objectives: Although many health care institutions believe that clinical peer review is vital for identifying and improving quality of care, peer review is perceived by many clinicians as variable and inherently punitive. Successful peer review requires institutional leadership and adoption of a just culture approach to investigating and determining accountability for medical errors that result in harm., Methods: We describe how an academic medical center implemented and adapted its clinical peer review processes to be consistent with just culture theory and provide a roadmap that other institutions may follow. Specific examples of peer review are highlighted to show how the process improved patient safety in the departments of emergency medicine, internal medicine, and pediatrics., Results: The most significant process improvement was shifting from a tradition of assigning letter grades of "A," "B," or "C" to determine whether preventable adverse events were caused by "human error," "at-risk behavior," or "reckless behavior." This categorization of human behaviors enabled patient safety officers within 3 departments to develop specific interventions to protect patients and enlist physician support for improving clinical systems., Conclusions: Each department's success was due to recognition of different patient and provider cultures that offer unique challenges. The transformation of peer review was a crucial first step to shift perceptions of peer review from a punitive to a constructive process intended to improve patient safety. Our experience with reengineering clinical peer review shows the importance of focusing on just culture as a key method to prevent patient harm., Competing Interests: The authors disclose no conflict of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Deep vein thrombosis is a major source of morbidity worldwide. For critical obstructions, catheter-directed thrombolytics are the frontline therapy to achieve vessel recanalization. Techniques that aid lytic therapy are under development to improve treatment efficacy and reduce procedure-related complications. Histotripsy is one such adjuvant under development that relies on focused ultrasound for in situ nucleation of bubble clouds. Prior studies have demonstrated synergistic effects for clot dissolution when histotripsy is combined with lytic therapy. The success of this combination approach is hypothesized to promote thrombolytic efficacy via two mechanisms: erythrocyte fractionation (hemolysis) and increased lytic activity (fibrinolysis). In this study, the contributions of hemolysis and fibrinolysis to clot degradation under histotripsy and a lytic were quantified with measurements of hemoglobin and D-dimer, respectively. A linear regression analysis was used to determine the relationship between hemoglobin, D-dimer, and the overall treatment efficacy (clot mass loss). A similar analysis was conducted to gauge the role of bubble activity, which was assessed with passive cavitation imaging, on hemolysis and fibrinolysis. Tabulation of these data demonstrated hemolysis and fibrinolysis contributed equally to clot mass loss. Furthermore, bubble cloud activity promoted the generation of hemoglobin and D-dimer in equal proportion. These studies indicate a multifactorial process for clot degradation under the action of histotripsy and a lytic therapy.

Bulk ultrasound ablation is a thermal therapy approach in which tissue is heated by unfocused or weakly focused sonication (average intensities on the order of 100 W/cm 2 ) to achieve coagulative necrosis within a few minutes exposure time. Assessing the role of bubble activity, including acoustic cavitation and tissue vaporization, in bulk ultrasound ablation may help in making bulk ultrasound ablation safer and more effective for clinical applications. Here, two series of ex vivo ablation trials were conducted to investigate the role of bubble activity and tissue vaporization in bulk ultrasound ablation. Fresh bovine liver tissue was ablated with unfocused, continuous-wave ultrasound using ultrasound image-ablate arrays sonicating at 31 W/cm 2 (0.9 MPa amplitude) for either 20 min at a frequency of 3.1 MHz or 10 min at 4.8 MHz. Tissue specimens were maintained at a static overpressure of either 0.52 or 1.2 MPa to suppress bubble activity and tissue vaporization or at atmospheric pressure for control groups. A passive cavitation detector was used to record subharmonic (1.55 or 2.4 MHz), broadband (1.2-1.5 MHz) and low-frequency (5-20 kHz) acoustic emissions. Treated tissue was stained with 2% triphenyl tetrazolium chloride to evaluate thermal lesion dimensions. Subharmonic emissions were significantly reduced in overpressure groups compared with control groups. Correlations observed between acoustic emissions and lesion dimensions were significant and positive for the 3.1-MHz series, but significant and negative for the 4.8-MHz series. The results indicate that for bulk ultrasound ablation, where both acoustic cavitation and tissue vaporization are possible, bubble activity can enhance ablation in the absence of tissue vaporization, but can reduce thermal lesion dimensions in the presence of vaporization., (Copyright © 2021 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

The EkoSonic endovascular system has been cleared by the U.S. Food and Drug Administration for the controlled and selective infusion of physician specified fluids, including thrombolytics, into the peripheral vasculature and the pulmonary arteries. The objective of this study was to explore whether this catheter technology could sustain cavitation nucleated by infused Definity, to support subsequent studies of ultrasound-mediated drug delivery to diseased arteries. The concentration and attenuation spectroscopy of Definity were assayed before and after infusion at 0.3, 2.0 and 4.0 mL/min through the EkoSonic catheter. PCI was used to map and quantify stable and inertial cavitation as a function of Definity concentration in a flow phantom mimicking the porcine femoral artery. The 2.0 mL/min infusion rate yielded the highest surviving Definity concentration and acoustic attenuation. Cavitation was sustained throughout each 15 ms ultrasound pulse, as well as throughout the 3 min infusion. These results demonstrate a potential pathway to use cavitation nucleation to promote drug delivery with the EkoSonic endovascular system., Competing Interests: Conflict of interest disclosure Boston Scientific provided the EkoSonic catheters and a driving unit. Curtis Genstler is an employee of Boston Scientific. Alexander S. Hannah is a former employee of Boston Scientific. The other authors have no conflicts of interest to disclose., (Copyright © 2020 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Adjuvant ultrasound at 2 MHz with or without an ultrasound contrast agent improves the rate of thrombus resolution by recombinant tissue plasminogen activator (rt-PA) in laboratory and clinical studies. A sub-megahertz approach can further expand this therapy to a subset of patients with an insufficient temporal bone window, improving efficacy in unselected patient populations. The aim of this study was to determine if a clinical ultrasound contrast agent (UCA), Definity, and 220 kHz pulsed ultrasound accelerated rt-PA thrombolysis in a preclinical animal model of vascular occlusion. The effect of Definity and ultrasound on thrombus clearance was first investigated in vitro and subsequently tested in a xenographic porcine cerebral thromboembolism model in vivo. Two different microcatheter designs (end-hole, multi-side-hole) were used to infuse rt-PA and Definity at the proximal edge or directly into clots, respectively. Sonothrombolysis with Definity increased clot mass loss relative to saline or rt-PA alone in vitro, only when rt-PA was administered directly into clots via a multi-side-hole microcatheter. Combined treatment with rt-PA, Definity, and ultrasound in vivo increased the rate of reperfusion up to 45 min faster than clots treated with rt-PA or saline. In this porcine cerebral thromboembolism model employing retracted human clots, 220 kHz ultrasound, in conjunction with Definity increased the probability of early successful reperfusion with rt-PA.

Aldehyde dehydrogenases (ALDHs) catalyze the conversion of various aliphatic and aromatic aldehydes into corresponding carboxylic acids. Traditionally considered as housekeeping enzymes, new biochemical roles are being identified for members of ALDH family. Recent work showed that AldA from the plant pathogen Pseudomonas syringae strain PtoDC3000 (PtoDC3000) functions as an indole-3-acetaldehyde dehydrogenase for the synthesis of indole-3-acetic acid (IAA). IAA produced by AldA allows the pathogen to suppress salicylic acid-mediated defenses in the model plant Arabidopsis thaliana. Here we present a biochemical and structural analysis of the AldA indole-3-acetaldehyde dehydrogenase from PtoDC3000. Site-directed mutants targeting the catalytic residues Cys302 and Glu267 resulted in a loss of enzymatic activity. The X-ray crystal structure of the catalytically inactive AldA C302A mutant in complex with IAA and NAD+ showed the cofactor adopting a conformation that differs from the previously reported structure of AldA. These structures suggest that NAD+ undergoes a conformational change during the AldA reaction mechanism similar to that reported for human ALDH. Site-directed mutagenesis of the IAA binding site indicates that changes in the active site surface reduces AldA activity; however, substitution of Phe169 with a tryptophan altered the substrate selectivity of the mutant to prefer octanal. The present study highlights the inherent biochemical versatility of members of the ALDH enzyme superfamily in P. syringae., (© 2020 The Author(s).)

Aldehyde dehydrogenases are versatile enzymes that serve a range of biochemical functions. Although traditionally considered metabolic housekeeping enzymes because of their ability to detoxify reactive aldehydes, like those generated from lipid peroxidation damage, the contributions of these enzymes to other biological processes are widespread. For example, the plant pathogen Pseudomonas syringae strain Pto DC3000 uses an indole-3-acetaldehyde dehydrogenase to synthesize the phytohormone indole-3-acetic acid to elude host responses. Here we investigate the biochemical function of AldC from Pto DC3000. Analysis of the substrate profile of AldC suggests that this enzyme functions as a long-chain aliphatic aldehyde dehydrogenase. The 2.5 Å resolution X-ray crystal of the AldC C291A mutant in a dead-end complex with octanal and NAD + reveals an apolar binding site primed for aliphatic aldehyde substrate recognition. Functional characterization of site-directed mutants targeting the substrate- and NAD(H)-binding sites identifies key residues in the active site for ligand interactions, including those in the "aromatic box" that define the aldehyde-binding site. Overall, this study provides molecular insight for understanding the evolution of the prokaryotic aldehyde dehydrogenase superfamily and their diversity of function., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Lee et al.)

In free-living and parasitic nematodes, the methylation of phosphoethanolamine to phosphocholine provides a key metabolite to sustain phospholipid biosynthesis for growth and development. Because the phosphoethanolamine methyltransferases (PMT) of nematodes are essential for normal growth and development, these enzymes are potential targets of inhibitor design. The pine wilt nematode (Bursaphelenchus xylophilus) causes extensive damage to trees used for lumber and paper in Asia. As a first step toward testing BxPMT1 as a potential nematicide target, we determined the 2.05 Å resolution x-ray crystal structure of the enzyme as a dead-end complex with phosphoethanolamine and S-adenosylhomocysteine. The three-dimensional structure of BxPMT1 served as a template for site-directed mutagenesis to probe the contribution of active site residues to catalysis and phosphoethanolamine binding using steady-state kinetic analysis. Biochemical analysis of the mutants identifies key residues on the β1d-α6 loop (W123F, M126I, and Y127F) and β1e-α7 loop (S155A, S160A, H170A, T178V, and Y180F) that form the phosphobase binding site and suggest that Tyr127 facilitates the methylation reaction in BxPMT1., Competing Interests: Declaration of Competing Interest The authors have no conflicting interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Therapeutic ultrasound strategies that harness the mechanical activity of cavitation nuclei for beneficial tissue bio-effects are actively under development. The mechanical oscillations of circulating microbubbles, the most widely investigated cavitation nuclei, which may also encapsulate or shield a therapeutic agent in the bloodstream, trigger and promote localized uptake. Oscillating microbubbles can create stresses either on nearby tissue or in surrounding fluid to enhance drug penetration and efficacy in the brain, spinal cord, vasculature, immune system, biofilm or tumors. This review summarizes recent investigations that have elucidated interactions of ultrasound and cavitation nuclei with cells, the treatment of tumors, immunotherapy, the blood-brain and blood-spinal cord barriers, sonothrombolysis, cardiovascular drug delivery and sonobactericide. In particular, an overview of salient ultrasound features, drug delivery vehicles, therapeutic transport routes and pre-clinical and clinical studies is provided. Successful implementation of ultrasound and cavitation nuclei-mediated drug delivery has the potential to change the way drugs are administered systemically, resulting in more effective therapeutics and less-invasive treatments., Competing Interests: Conflict of interest disclosure The authors declare no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Phytochemical diversity is thought to result from coevolutionary cycles as specialization in herbivores imposes diversifying selection on plant chemical defenses. Plants in the speciose genus Erysimum (Brassicaceae) produce both ancestral glucosinolates and evolutionarily novel cardenolides as defenses. Here we test macroevolutionary hypotheses on co-expression, co-regulation, and diversification of these potentially redundant defenses across this genus. We sequenced and assembled the genome of E. cheiranthoides and foliar transcriptomes of 47 additional Erysimum species to construct a phylogeny from 9868 orthologous genes, revealing several geographic clades but also high levels of gene discordance. Concentrations, inducibility, and diversity of the two defenses varied independently among species, with no evidence for trade-offs. Closely related, geographically co-occurring species shared similar cardenolide traits, but not glucosinolate traits, likely as a result of specific selective pressures acting on each defense. Ancestral and novel chemical defenses in Erysimum thus appear to provide complementary rather than redundant functions., Competing Interests: TZ, SS, AP, MM, HA, MM, TY, CH, PK, ME, GP, JG, FP, CM, JP, LM, GJ No competing interests declared, (© 2020, Züst et al.)

Phytoalexins play a pivotal role in plant-pathogen interactions. Whereas leaves of rice (Oryza sativa) cultivar Nipponbare predominantly accumulated the phytoalexin sakuranetin after jasmonic acid induction, only very low amounts accumulated in the Kasalath cultivar. Sakuranetin is synthesized from naringenin by naringenin 7-O-methyltransferase (NOMT). Analysis of chromosome segment substitution lines and backcrossed inbred lines suggested that NOMT is the underlying cause of differential phytoalexin accumulation between Nipponbare and Kasalath. Indeed, both NOMT expression and NOMT enzymatic activity are lower in Kasalath than in Nipponbare. We identified a proline to threonine substitution in Kasalath relative to Nipponbare NOMT as the main cause of the lower enzymatic activity. Expanding this analysis to rice cultivars with varying amounts of sakuranetin collected from around the world showed that NOMT induction is correlated with sakuranetin accumulation. In bioassays with Pyricularia oryzae, Gibberella fujikuroi, Bipolaris oryzae, Burkholderia glumae, Xanthomonas oryzae, Erwinia chrysanthemi, Pseudomonas syringae, and Acidovorax avenae, naringenin was more effective against bacterial pathogens and sakuranetin was more effective against fungal pathogens. Therefore, the relative amounts of naringenin and sakuranetin may provide protection against specific pathogen profiles in different rice-growing environments. In a dendrogram of NOMT genes, those from low-sakuranetin-accumulating cultivars formed at least two clusters, only one of which involves the proline to threonine mutation, suggesting that the low sakuranetin chemotype was acquired more than once in cultivated rice. Strains of the wild rice species Oryza rufipogon also exhibited differential sakuranetin accumulation, indicating that this metabolic diversity predates rice domestication., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Although primarily known as an ablative modality, histotripsy can increase the efficacy of lytic therapy in a retracted venous clot model. Bubble cloud oscillations are the primary mechanism of action for histotripsy, and the type of bubble activity is dependent on the pulse duration. A retracted human venous clot model was perfused with and without the thrombolytic recombinant tissue plasminogen activator (rt-PA). The clot was exposed to histotripsy pulses of single- or five-cycle duration and peak negative pressures of 0-30 MPa. Bubble activity within the clot was monitored via passive cavitation imaging. The combination of histotripsy and rt-PA was more efficacious than rt-PA alone for single- and five-cycle pulses with peak negative pressures of 25 and 20 MPa, respectively. For both excitation schemes, the detected acoustic emissions correlated with the degree of thrombolytic efficacy. These results indicate that rt-PA and single- or multicycle histotripsy pulses enhance thrombolytic therapy., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Ultrasound has been developed as both a diagnostic tool and a potent promoter of beneficial bio-effects for the treatment of chronic bacterial infections. Bacterial infections, especially those involving biofilm on implants, indwelling catheters and heart valves, affect millions of people each year, and many deaths occur as a consequence. Exposure of microbubbles or droplets to ultrasound can directly affect bacteria and enhance the efficacy of antibiotics or other therapeutics, which we have termed sonobactericide. This review summarizes investigations that have provided evidence for ultrasound-activated microbubble or droplet treatment of bacteria and biofilm. In particular, we review the types of bacteria and therapeutics used for treatment and the in vitro and pre-clinical experimental setups employed in sonobactericide research. Mechanisms for ultrasound enhancement of sonobactericide, with a special emphasis on acoustic cavitation and radiation force, are reviewed, and the potential for clinical translation is discussed., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The global pandemic of antibiotic resistance is an ever-burgeoning public health challenge, motivating the development of adjunct bactericidal therapies. Nitric oxide (NO) is a potent bioactive gas that induces a variety of therapeutic effects, including bactericidal and biofilm dispersion properties. The short half-life, high reactivity, and rapid diffusivity of NO make therapeutic delivery challenging. The goal of this work was to characterize NO-loaded microbubbles (MB) stabilized with a lipid shell and to assess the feasibility of antibacterial therapy in vitro . MB were loaded with either NO alone (NO-MB) or with NO and octafluoropropane (NO-OFP-MB) (9:1 v/v and 1:1 v/v). The size distribution and acoustic attenuation coefficient of NO-MB and NO-OFP-MB were measured. Ultrasound-triggered release of the encapsulated gas payload was demonstrated with 3-MHz pulsed Doppler ultrasound. An amperometric microelectrode sensor was used to measure NO concentration released from the MB and compared to an NO-OFP-saturated solution. The effect of NO delivery on the viability of planktonic (free living) Staphylococcus aureus (SA) USA 300, a methicillin-resistant strain, was evaluated in a 96 well-plate format. The co-encapsulation of NO with OFP increased the total volume and attenuation coefficient of MB. The NO-OFP-MB were destroyed with a clinical ultrasound scanner with an output of 2.48 MPa peak negative pressure ( in situ MI of 1.34) but maintained their echogenicity when exposed to 0.02 MPa peak negative pressure ( in situ MI of 0.01. The NO dose in NO-MB and NO-OFP-MB was more than 2-fold higher than the NO-OFP-saturated solution. Delivery of NO-OFP-MB increased bactericidal efficacy compared to the NO-OFP-saturated solution or air and OFP-loaded MB. These results suggest that encapsulation of NO with OFP in lipid-shelled MB enhances payload delivery. Furthermore, these studies demonstrate the feasibility and limitations of NO-OFP-MB for antibacterial applications., (Copyright © 2020 Lafond, Shekhar, Panmanee, Collins, Palaniappan, McDaniel, Hassett and Holland.)

Late in-stent restenosis remains a significant problem. Bare-metal stents were implanted into peripheral arteries in miniature swine, followed by direct intra-arterial infusion of nitric oxide-loaded echogenic liposomes (ELIPs) and anti-intercellular adhesion molecule-1 conjugated ELIPs loaded with pioglitazone exposed to an endovascular catheter with an ultrasonic core. Ultrasound-facilitated delivery of ELIP formulations into stented peripheral arteries attenuated neointimal growth. Local atheroma-targeted, ultrasound-triggered delivery of nitric oxide and pioglitazone, an anti-inflammatory peroxisome proliferator-activated receptor-γ agonist, into stented arteries has the potential to stabilize stent-induced neointimal growth and obviate the need for long-term antiplatelet therapy., (© 2020 The Authors.)

To modulate responses to developmental or environmental cues, plants use Gretchen Hagen 3 (GH3) acyl acid amido synthetases to conjugate an amino acid to a plant hormone, a reaction that regulates free hormone concentration and downstream responses. The model plant Arabidopsis thaliana has 19 GH3 proteins, of which 8 have confirmed biochemical functions. One Brassicaceae-specific clade of GH3 proteins was predicted to use benzoate as a substrate and includes AtGH3.7 and AtGH3.12/PBS3. Previously identified as a 4-hydroxybenzoic acid-glutamate synthetase, AtGH3.12/PBS3 influences pathogen defense responses through salicylic acid. Recent work has shown that AtGH3.12/PBS3 uses isochorismate as a substrate, forming an isochorismate-glutamate conjugate that converts into salicylic acid. Here, we show that AtGH3.7 and AtGH3.12/PBS3 can also conjugate chorismate to cysteine and glutamate, which act as precursors to aromatic amino acids and salicylic acid, respectively. The X-ray crystal structure of AtGH3.12/PBS3 in complex with AMP and chorismate at 1.94 Å resolution, along with site-directed mutagenesis, revealed how the active site potentially accommodates this substrate. Examination of Arabidopsis knockout lines indicated that the gh3.7 mutants do not alter growth and showed no increased susceptibility to the pathogen Pseudomonas syringae , unlike gh3.12 mutants, which were more susceptible than WT plants, as was the gh3.7 / gh3.12 double mutant. The findings of our study suggest that GH3 proteins can use metabolic precursors of aromatic amino acids as substrates., (© 2019 Holland et al.)

Acoustic droplet vaporization (ADV) has been shown to reduce the partial pressure of oxygen (PO 2 ) in a fluid. The goals of this study were three-fold: 1) to determine the ADV pressure amplitude threshold in fluids that had physiologically relevant values for surface tension, protein concentration, and viscosity; 2) to assess whether these parameters and fluid mixing affect ADV-mediated PO 2 reduction; and 3) to assess the feasibility of ADV-mediated PO 2 reduction in plasma and whole blood. In vitro ADV experiments were conducted using perfluoropentane droplets (number density: 5 × 10 6  ± 0.2 × 10 6 /mL) dispersed in fluids (saline, polyvinylpyrrolidone solutions, porcine plasma, or porcine whole blood) that had a physiological range of surface tensions (62-68 mN/m), protein concentrations (0 and 68.7 mg/mL), and viscosities (0.7-4 cP). Droplets were exposed to pulsed ultrasound (5 MHz, 4.25 MPa peak negative pressure) while passing through a 37 °C flow system with inline PO 2 sensors. In select experiments, the fluid also passed through mixing channels after ultrasound exposure. Our results revealed that the ADV pressure thresholds were the same for all fluids. Surface tension and protein concentration had no effect on PO 2 reduction. Increasing viscosity attenuated PO 2 reduction. However, the attenuated effect was absent after fluid mixing. Furthermore, ADV-mediated PO 2 reduction in whole blood (30.8 ± 3.2 mmHg) was less than that in a polyvinylpyrrolidone solution (40.2 ± 2.1 mmHg) with equal viscosity. These findings should be considered when planning clinical studies of ADV-mediated PO 2 reduction and other biomedical applications of ADV., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ultrasound-enhanced recombinant tissue plasminogen activator (rt-PA) thrombolysis is under development as an adjuvant to ischemic stroke therapy. The goal of this study was to design a pulsed ultrasound (US) exposure scheme that reduced intracranial constructive interference and tissue heating, and maintained thrombolytic efficacy relative to continuous wave (CW) insonation. Three 220 kHz US schemes were evaluated, two pulsed insonation schemes (15 cycles, 68 µs pulse duration, 33% or 62.5% duty cycle) and an intermittent CW insonation scheme (50 s active, 30 s quiescent) over a 30-min treatment period. An in silico study using a finite-difference model of transcranial US propagation was performed to estimate the intracranial acoustic field and temperature rise in the skull for each insonation scheme. In vitro measurements with flow were performed to assess thrombolysis using time-lapse microscopy. Intracranial constructive interference was not reduced with pulsed US using a pulse length of 15 cycles compared to intermittent CW US. The 33.3% duty cycle pulsed US scheme reduced heating in the temporal bone as much as 60% relative to the intermittent CW scheme. All insonation schemes promoted sustained stable cavitation in vitro and augmented thrombolysis compared to rt-PA alone (p   <  0.05). Ultraharmonic (UH) and harmonic cumulative energy over a 30 min treatment period was significantly higher (p   <  0.05) for the intermittent CW US scheme compared to either pulsed US scheme. Despite the difference in cavitation emissions, no difference was observed in the clot lysis between the three US schemes. These findings demonstrate that a 33.3% duty cycle pulsed US scheme with a 15-cycle burst can reduce bone heating and achieve equivalent thrombolytic efficacy as an intermittent CW scheme.

The development of adjuvant techniques to improve thrombolytic efficacy is important for advancing ischemic stroke therapy. We characterized octafluoropropane and recombinant tissue plasminogen activator (rt-PA)-loaded echogenic liposomes (OFP t-ELIP) using differential interference and fluorescence microscopy, attenuation spectroscopy, and electrozone sensing. The loading of rt-PA in OFP t-ELIP was assessed using spectrophotometry. Further, it was tested whether the agent shields rt-PA against degradation by plasminogen activator inhibitor-1 (PAI-1). An in vitro system was used to assess whether ultrasound (US) combined with either Definity or OFP t-ELIP enhances rt-PA thrombolysis. Human whole blood clots were mounted in a flow system and visualized using an inverted microscope. The perfusate consisted of either (1) plasma alone, (2) rt-PA, (3) OFP t-ELIP, (4) rt-PA and US, (5) OFP t-ELIP and US, (6) Definity and US, or (7) rt-PA, Definity, and US (n = 16 clots per group). An intermittent US insonation scheme was employed (220 kHz frequency, and 0.44 MPa peak-to-peak pressures) for 30 min. Microscopic imaging revealed that OFP t-ELIP included a variety of structures such as liposomes (with and without gas) and lipid-shelled microbubbles. OFP t-ELIP preserved up to 76% of rt-PA activity in the presence of PAI-1, whereas only 24% activity was preserved for unencapsulated rt-PA. The use of US with rt-PA and Definity enhanced lytic efficacy (p < 0.05) relative to rt-PA alone. US combined with OFP t-ELIP enhanced lysis over OFP t-ELIP alone (p < 0.01). These results demonstrate that ultrasound combined with Definity or OFP t-ELIP can enhance the lytic activity relative to rt-PA or OFP t-ELIP alone, respectively.