Carbon Monoxide Releasing Molecule A1 Reduces Myocardial Damage After Acute Myocardial Infarction in a Porcine Model.

ABSTRACT: Infarct size is a major determinant of outcomes after acute myocardial infarction (AMI). Carbon monoxide-releasing molecules (CORMs), which deliver nanomolar concentrations of carbon monoxide to tissues, have been shown to reduce infarct size in rodents. We evaluated efficacy and safety of CORM-A1 to reduce infarct size in a clinically relevant porcine model of AMI. We induced AMI in Yorkshire White pigs by inflating a coronary angioplasty balloon to completely occlude the left anterior descending artery for 60 minutes, followed by deflation of the balloon to mimic reperfusion. Fifteen minutes after balloon occlusion, animals were given an infusion of 4.27 mM CORM-A1 (n = 7) or sodium borate control (n = 6) over 60 minutes. Infarct size, cardiac biomarkers, ejection fraction, and hepatic and renal function were compared amongst the groups. Immunohistochemical analyses were performed to compare inflammation, cell proliferation, and apoptosis between the groups. CORM-A1-treated animals had significant reduction in absolute infarct area (158 ± 16 vs. 510 ± 91 mm2, P < 0.001) and infarct area corrected for area at risk (24.8% ± 2.6% vs. 45.2% ± 4.0%, P < 0.0001). Biochemical markers of myocardial injury also tended to be lower and left ventricular function tended to recover better in the CORM-A1 treated group. There was no evidence of hepatic or renal toxicity with the doses used. The cardioprotective effects of CORM-A1 were associated with a significant reduction in cell proliferation and inflammation. CORM-A1 reduces infarct size and improves left ventricular remodeling and function in a porcine model of reperfused MI by a reduction in inflammation. These potential cardioprotective effects of CORMs warrant further translational investigations.

Automating neurosurgical tumor resection surgery: Volumetric laser ablation of cadaveric porcine brain with integrated surface mapping.

OBJECTIVE: Current surgical instruments for soft tissue resection including neurosurgical procedures rely on the accuracy and precision of the human operator and are fundamentally constrained by the human hand. Automated surgical action with the integration of intraoperative data sources can enable highly accurate and fast tissue manipulation using laser ablation. This study presents the first experiments with a prototype designed for automated tumor resection via laser ablation. We demonstrate targeted soft tissue resection in porcine brain with an integrated device that combines 3D scanning capabilities with a steerable surgical laser and discuss implications for future automated robotic neurosurgical procedures. STUDY DESIGN AND METHODS: A device consisting of a two-axis galvanometer for steering a cutting laser and a 3D surface profiler is used to perform volumetric removal of tissue of ex vivo porcine brain. Three-dimensional surface profiles are gathered between cuts and used to estimate ablation rate. RESULTS: Volumetric ablation of porcine brain tissue is performed and subsequently surface profiled. The average ablation rates across the area cutting areas were 2.6 mm3 /s and 3.7 mm3 /s for the initial and subsequent cuts, respectively. A Kruskal-Wallis and post-hoc Tukey test show statistical significance between the initial and subsequent cuts. Accuracy between cuts when benchmarked against a human surgeon varied from 47 to 88%. CONCLUSION: A feed-forward volumetric resection is demonstrated with sensing and cutting housed within a single device, thereby opening the potential for automated soft tissue resection as necessary during the surgical removal of pathologic tissues. High variance around target cut depths motivates future work in developing a closed-loop ablation tool as well as characterization of laser-tissue interactions for predictive modelling. Objective Lasers Surg. 50:1017-1024, 2018. © 2018 Wiley Periodicals, Inc.

Activation of protein kinase PKR requires dimerization-induced cis-phosphorylation within the activation loop.

Protein kinase R (PKR) functions in a plethora of cellular processes, including viral and cellular stress responses, by phosphorylating the translation initiation factor eIF2α. The minimum requirements for PKR function are homodimerization of its kinase and RNA-binding domains, and autophosphorylation at the residue Thr-446 in a flexible loop called the activation loop. We investigated the interdependence between dimerization and Thr-446 autophosphorylation using the yeast Saccharomyces cerevisiae model system. We showed that an engineered PKR that bypassed the need for Thr-446 autophosphorylation (PKR(T446∼P)-bypass mutant) could function without a key residue (Asp-266 or Tyr-323) that is essential for PKR dimerization, suggesting that dimerization precedes and stimulates activation loop autophosphorylation. We also showed that the PKR(T446∼P)-bypass mutant was able to phosphorylate eIF2α even without its RNA-binding domains. These two significant findings reveal that PKR dimerization and activation loop autophosphorylation are mutually exclusive yet interdependent processes. Also, we provide evidence that Thr-446 autophosphorylation during PKR activation occurs in a cis mechanism following dimerization.

Carbon monoxide-releasing molecule-3 (CORM-3; Ru(CO)3Cl(glycinate)) as a tool to study the concerted effects of carbon monoxide and nitric oxide on bacterial flavohemoglobin Hmp: applications and pitfalls.

CO and NO are small toxic gaseous molecules that play pivotal roles in biology as gasotransmitters. During bacterial infection, NO, produced by the host via the inducible NO synthase, exerts critical antibacterial effects while CO, generated by heme oxygenases, enhances phagocytosis of macrophages. In Escherichia coli, other bacteria and fungi, the flavohemoglobin Hmp is the most important detoxification mechanism converting NO and O2 to the ion nitrate (NO3(-)). The protoheme of Hmp binds not only O2 and NO, but also CO so that this ligand is expected to be an inhibitor of NO detoxification in vivo and in vitro. CORM-3 (Ru(CO)(3)Cl(glycinate)) is a metal carbonyl compound extensively used and recently shown to have potent antibacterial properties. In this study, attenuation of the NO resistance of E. coli by CORM-3 is demonstrated in vivo. However, polarographic measurements showed that CO gas, but not CORM-3, produced inhibition of the NO detoxification activity of Hmp in vitro. Nevertheless, CO release from CORM-3 in the presence of soluble cellular compounds is demonstrated by formation of carboxy-Hmp. We show that the inability of CORM-3 to inhibit the activity of purified Hmp is due to slow release of CO in protein solutions alone i.e. when sodium dithionite, widely used in previous studies of CO release from CORM-3, is excluded. Finally, we measure intracellular CO released from CORM-3 by following the formation of carboxy-Hmp in respiring cells. CORM-3 is a tool to explore the concerted effects of CO and NO in vivo.

Cytochrome bd-I in Escherichia coli is less sensitive than cytochromes bd-II or bo'' to inhibition by the carbon monoxide-releasing molecule, CORM-3: N-acetylcysteine reduces CO-RM uptake and inhibition of respiration.

BACKGROUND: CO-releasing molecules (CO-RMs) are potential therapeutic agents, able to deliver CO - a critical gasotransmitter - in biological environments. CO-RMs are also effective antimicrobial agents; although the mechanisms of action are poorly defined, haem-containing terminal oxidases are primary targets. Nevertheless, it is clear from several studies that the effects of CO-RMs on biological systems are frequently not adequately explained by the release of CO: CO-RMs are generally more potent inhibitors than is CO gas and other effects of the molecules are evident. METHODS: Because sensitivity to CO-RMs cannot be predicted by sensitivity to CO gas, we assess the differential susceptibilities of strains, each expressing only one of the three terminal oxidases of E. coli - cytochrome bd-I, cytochrome bd-II and cytochrome bo', to inhibition by CORM-3. We present the first sensitive measurement of the oxygen affinity of cytochrome bd-II (Km 0.24µM) employing globin deoxygenation. Finally, we investigate the way(s) in which thiol compounds abolish the inhibitory effects of CORM-2 and CORM-3 on respiration, growth and viability, a phenomenon that is well documented, but poorly understood. RESULTS: We show that a strain expressing cytochrome bd-I as the sole oxidase is least susceptible to inhibition by CORM-3 in its growth and respiration of both intact cells and membranes. Growth studies show that cytochrome bd-II has similar CORM-3 sensitivity to cytochrome bo'. Cytochromes bo' and bd-II also have considerably lower affinities for oxygen than bd-I. We show that the ability of N-acetylcysteine to abrogate the toxic effects of CO-RMs is not attributable to its antioxidant effects, or prevention of CO targeting to the oxidases, but may be largely due to the inhibition of CO-RM uptake by bacterial cells. CONCLUSIONS: A strain expressing cytochrome bd-I as the sole terminal oxidase is least susceptible to inhibition by CORM-3. N-acetylcysteine is a potent inhibitor of CO-RM uptake by E. coli. GENERAL SIGNIFICANCE: Rational design and exploitation of CO-RMs require a fundamental understanding of their activity. CO and CO-RMs have multifaceted effects on mammalian and microbial cells; here we show that the quinol oxidases of E. coli are differentially sensitive to CORM-3. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Co-circulation of West Nile virus variants, Arizona, USA, 2010.

Molecular analysis of West Nile virus (WNV) isolates obtained during a 2010 outbreak in Maricopa County, Arizona, USA, demonstrated co-circulation of 3 distinct genetic variants, including strains with novel envelope protein mutations. These results highlight the continuing evolution of WNV in North America and the current complexity of WNV dispersal and transmission.

Interaction of the carbon monoxide-releasing molecule Ru(CO)3Cl(glycinate) (CORM-3) with Salmonella enterica serovar Typhimurium: in situ measurements of carbon monoxide binding by integrating cavity dual-beam spectrophotometry.

Carbon monoxide (CO) is a toxic gas that binds to haems, but also plays critical signalling and cytoprotective roles in mammalian systems; despite problems associated with systemic delivery by inhalation of the gas, it may be employed therapeutically. CO delivered to cells and tissues by CO-releasing molecules (CO-RMs) has beneficial and toxic effects not mimicked by CO gas; CO-RMs are also attractive candidates as novel antimicrobial agents. Salmonella enterica serovar Typhimurium is an enteropathogen causing gastroenteritis in humans. Recent studies have implicated haem oxygenase-1 (HO-1), the protein that catalyses the degradation of haem into biliverdin, free iron and CO, in the host immune response to Salmonella infection. In several studies, CO administration via CO-RMs elicited many of the protective roles of HO-1 induction and so we investigated the effects of a well-characterized water-soluble CO-RM, Ru(CO)3Cl(glycinate) (CORM-3), on Salmonella. CORM-3 exhibits toxic effects at concentrations significantly lower than those reported to cause toxicity to RAW 264.7 macrophages. We demonstrated here, through oxyhaemoglobin assays, that CORM-3 did not release CO spontaneously in phosphate buffer, buffered minimal medium or very rich medium. CORM-3 was, however, accumulated to high levels intracellularly (as shown by inductively coupled plasma MS) and released CO inside cells. Using growing Salmonella cultures without prior concentration, we showed for the first time that sensitive dual-beam integrating cavity absorption spectrophotometry can detect directly the CO released from CORM-3 binding in real-time to haems of the bacterial electron transport chain. The toxic effects of CO-RMs suggested potential applications as adjuvants to antibiotics in antimicrobial therapy.

Continued evolution of West Nile virus, Houston, Texas, USA, 2002-2012.

We investigated the genetics and evolution of West Nile virus (WNV) since initial detection in the United States in 1999 on the basis of continual surveillance studies in the Houston, Texas, USA, metropolitan area (Harris County) as a surrogate model for WNV evolution on a national scale. Full-length genomic sequencing of 14 novel 2010-2012 WNV isolates collected from resident birds in Harris County demonstrates emergence of 4 independent genetic groups distinct from historical strains circulating in the greater Houston region since 2002. Phylogenetic and geospatial analyses of the 2012 WNV isolates indicate closer genetic relationship with 2003-2006 Harris County isolates than more recent 2007-2011 isolates. Inferred monophyletic relationships of these groups with several 2006-2009 northeastern US isolates supports potential introduction of a novel WNV strain in Texas since 2010. These results emphasize the need to maintain WNV surveillance activities to better understand WNV transmission dynamics in the United States.

Vasorelaxing effects and inhibition of nitric oxide in macrophages by new iron-containing carbon monoxide-releasing molecules (CO-RMs).

Carbon monoxide-releasing molecules (CO-RMs) are a class of organometallo carbonyl complexes capable of delivering controlled quantities of CO gas to cells and tissues thus exerting a broad spectrum of pharmacological effects. Here we report on the chemical synthesis, CO releasing properties, cytotoxicity profile and pharmacological activities of four novel structurally related iron-allyl carbonyls. The major difference among the new CO-RMs tested was that three compounds (CORM-307, CORM-308 and CORM-314) were soluble in dimethylsulfoxide (DMSO), whereas a fourth one (CORM-319) was rendered water-soluble by reacting the iron-carbonyl with hydrogen tetrafluoroborate. We found that despite the fact all compounds liberated CO, CO-RMs soluble in DMSO caused a more pronounced toxic effect both in vascular and inflammatory cells as well as in isolated vessels. More specifically, iron carbonyls soluble in DMSO released CO with a fast kinetic and displayed a marked cytotoxic effect in smooth muscle cells and RAW 247.6 macrophages despite exerting a rapid and pronounced vasorelaxation ex vivo. In contrast, CORM-319 that is soluble in water and liberated CO with a slower rate, preserved smooth muscle cell viability, relaxed aortic tissue and exerted a significant anti-inflammatory effect in macrophages challenged with endotoxin. These data suggest that iron carbonyls can be used as scaffolds for the design and synthesis of pharmacologically active CO-RMs and indicate that increasing water solubility and controlling the rate of CO release are important parameters for limiting their potential toxic effects.

Antithrombotic properties of water-soluble carbon monoxide-releasing molecules.

OBJECTIVE: We compared the antithrombotic effects in vivo of 2 chemically different carbon monoxide-releasing molecules (CORM-A1 and CORM-3) on arterial and venous thrombus formation and on hemostatic parameters such as platelet activation, coagulation, and fibrinolysis. The hypotensive response to CORMs and their effects on whole blood gas analysis and blood cell count were also examined. METHODS AND RESULTS: CORM-A1 (10-30 µmol/kg, i.v.), in a dose-dependent fashion, significantly decreased weight of electrically induced thrombus in rats, whereas CORM-3 inhibited thrombosis only at the highest dose used (30 µmol/kg). CORM-A1 showed a direct and stronger inhibition of platelet aggregation than CORM-3 in healthy rats, both in vitro and in vivo. The antiaggregatory effect of CORM-A1, but not CORM-3, correlated positively with weight of the thrombus. Concentration of active plasminogen activator inhibitor-1 in plasma also decreased in response to CORM-A1, but not to CORM-3. Neither CORM-A1 nor CORM-3 had an effect on plasma concentration of active tissue plasminogen activator. CORM-3, but not CORM-A1, decreased the concentration of fibrinogen, fibrin generation, and prolonged prothrombin time. Similarly, laser-induced venous thrombosis observed intravitally via confocal system in green fluorescent protein mice was significantly decreased by CORMs. Although both CORM-A1 and CORM-3 (30 µmol/kg) decreased platelets accumulation in thrombus, only CORM-A1 (3-30 µmol/kg) inhibited platelet activation to phosphatidylserine on their surface. CONCLUSIONS: CORM-3 and CORM-A1 inhibited thrombosis in vivo, however CORM-A1, which slowly releases carbon monoxide, and displayed a relatively weak hypotensive effect had a more pronounced antithrombotic effect associated with a stronger inhibition of platelet aggregation associated with a decrease in active plasminogen activator inhibitor-1 concentration. In contrast, the fast CO releaser CORM-3 that displayed a more pronounced hypotensive effect inhibited thrombosis primarily through a decrease in fibrin generation, but had no direct influence on platelet aggregation and fibrynolysis.

Imprinted Anti-Hemagglutinin and Anti-Neuraminidase Antibody Responses after Childhood Infections of A(H1N1) and A(H1N1)pdm09 Influenza Viruses.

Immune imprinting is a driver known to shape the anti-hemagglutinin (HA) antibody landscape of individuals born within the same birth cohort. With the HA and neuraminidase (NA) proteins evolving at different rates under immune selection pressures, anti-HA and anti-NA antibody responses since childhood influenza virus infections have not been evaluated in parallel at the individual level. This is partly due to the limited knowledge of changes in NA antigenicity, as seasonal influenza vaccines have focused on generating neutralizing anti-HA antibodies against HA antigenic variants. Here, we systematically characterized the NA antigenic variants of seasonal A(H1N1) viruses from 1977 to 1991 and completed the antigenic profile of N1 NAs from 1977 to 2015. We identified that NA proteins of A/USSR/90/77, A/Singapore/06/86, and A/Texas/36/91 were antigenically distinct and mapped N386K as a key determinant of the NA antigenic change from A/USSR/90/77 to A/Singapore/06/86. With comprehensive panels of HA and NA antigenic variants of A(H1N1) and A(H1N1)pdm09 viruses, we determined hemagglutinin inhibition (HI) and neuraminidase inhibition (NI) antibodies from 130 subjects born between 1950 and 2015. Age-dependent imprinting was observed for both anti-HA and anti-NA antibodies, with the peak HI and NI titers predominantly detected from subjects at 4 to 12 years old during the year of initial virus isolation, except the age-independent anti-HA antibody response against A(H1N1)pdm09 viruses. More participants possessed antibodies that reacted to multiple antigenically distinct NA proteins than those with antibodies that reacted to multiple antigenically distinct HA proteins. Our results support the need to include NA proteins in seasonal influenza vaccine preparations. IMPORTANCE Seasonal influenza vaccines have aimed to generate neutralizing anti-HA antibodies for protection since licensure. More recently, anti-NA antibodies have been established as an additional correlate of protection. While HA and NA antigenic changes occurred discordantly, the anti-HA and anti-NA antibody profiles have rarely been analyzed in parallel at the individual level, due to the limited knowledge on NA antigenic changes. By characterizing NA antigenic changes of A(H1N1) viruses, we determined the anti-HA and anti-NA antibody landscape against antigenically distinct A(H1N1) and A(H1N1)pdm09 viruses using sera of 130 subjects born between 1950 and 2015. We observed age-dependent imprinting of both anti-HA and anti-NA antibodies against strains circulated during the first decade of life. A total of 67.7% (88/130) and 90% (117/130) of participants developed cross-reactive antibodies to multiple HA and NA antigens at titers ≥1:40. With slower NA antigenic changes and cross-reactive anti-NA antibody responses, including NA protein in influenza vaccine preparation may enhance vaccine efficacy.

Cross-neutralization and viral fitness of SARS-CoV-2 Omicron sublineages.

The rapid evolution of SARS-CoV-2 Omicron sublineages mandates a better understanding of viral replication and cross-neutralization among these sublineages. Here we used K18-hACE2 mice and primary human airway cultures to examine the viral fitness and antigenic relationship among Omicron sublineages. In both K18-hACE2 mice and human airway cultures, Omicron sublineages exhibited a replication order of BA.5 ≥ BA.2 ≥ BA.2.12.1 > BA.1; no difference in body weight loss was observed among different sublineage-infected mice. The BA.1-, BA.2-, BA.2.12.1-, and BA.5-infected mice developed distinguishable cross-neutralizations against Omicron sublineages, but exhibited little neutralization against the index virus (i.e. USA-WA1/2020) or the Delta variant. Surprisingly, the BA.5-infected mice developed higher neutralization activity against heterologous BA.2 and BA.2.12.1 than that against homologous BA.5; serum neutralizing titres did not always correlate with viral replication levels in infected animals. Our results revealed a distinct antigenic cartography of Omicron sublineages and support the bivalent vaccine approach.

Sulfite species enhance carbon monoxide release from CO-releasing molecules: implications for the deoxymyoglobin assay of activity.

Carbon monoxide-releasing molecules (CO-RMs) emulate the beneficial (e.g., anti-inflammatory) effects of CO in biology. CO release from CO-RMs is routinely determined in the presence of reduced deoxy-myoglobin by measuring the formation of carboxy-myoglobin (Mb-CO). Previous studies have highlighted discrepancies between the apparent CO release rates of some CO-RMs established using this assay versus other experimental data where a slower or more complex mechanism of release is suggested. It has been hypothesized that some CO-RMs require a CO acceptor, believed to be reduced myoglobin in Mb-CO assays, in order to facilitate the release of CO. Here, we show, for the first time, that CO is not liberated from the ruthenium (Ru)-based [Ru(CO)(3)Cl(2)](2) (CORM-2) and [Ru(CO)(3)Cl(glycinate)] (CORM-3) at an appreciable rate in the presence of reduced myoglobin alone. Rather, we confirm that it is the reducing agent sodium dithionite that facilitates release of CO from these CO-RMs. Other sulfite compounds, namely sodium sulfite and potassium metabisulfite, also promote the liberation of CO from CORM-3. We describe an alternative oxy-hemoglobin assay that eliminates dithionite and suggest that the efficacy of CO-RMs results from intracellular interactions with anions that facilitate CO delivery to therapeutic targets.

"Every organ, every time" attitude teamed up with SoftMD results in successful lung transplant from a 72-year-old donor.

Since 1998, lung transplants have increased almost 75%, making lung transplant the fastest growing form of organ transplant in the United States. However, the supply of transplantable lungs continues to fall short of the demand. Strategies for expanding the lung donor pool by increasing the number of older donor lungs used has been effective, although these donors do not meet typical clinical selection criteria based on age. In these older donors, effective communication of in-depth donor information is necessary to place and transplant lungs successfully. The following case study illustrates how an "every organ, every time" attitude combined with a technique to communicate clinical information resulted in the successful transplant of 72-year-old donor lungs into a bilateral lung recipient. Since the case outlined in this study, the organ procurement organization has successfully recovered and transplanted an additional 2 lungs from a 74-year-old and 1 lung from a 76-year-old, 2 of the oldest lung donors in the United States. This case demonstrates that although many older donor lungs are deemed unsuitable by clinical selection criteria, an extended criteria population offers an untapped resource for donor organs.

Medical students' perceptions of understanding skills needed in treating cardiac patients through simulation-based encounters in a clinical skills curriculum.

CONTEXT: Simulation-based education can enhance medical students' understanding of clinical concepts as they learn the key elements needed to treat patients with various medical conditions. The integration of simulation programs into medical school curricula increases students' exposure to this type of learning. OBJECTIVES: To determine the effectiveness of simulation activities on medical students' perceptions of understanding cardiac rhythm identification and the pharmacology skills necessary to manage a stable patient with cardiac arrhythmia. METHODS: A retrospective secondary data analysis was conducted utilizing a quasi-experimental one-group pretest/posttest study of a convenience sample of 159 second-year medical students in the southeastern United States during the 2017-2018 academic year. Because this was a one-group pretest/posttest study, only second-year medical students whose data could be matched were included. A 5-point Likert scale, previously developed as part of the clinical skills course, was utilized to collect ordinal understanding of cardiac rhythm identification and the pharmacologic management of a stable patient with cardiac arrhythmia. The intervention was an 18-min simulated patient encounter involving a high-fidelity mannequin (SimMan 3G or Essential) with supraventricular tachycardia. RESULTS: Of the 159 students, the number of students who reported a good perception of understanding of cardiac rhythm identification presimulation activity increased from 44.0% (70) to 52.2% (83) postsimulation activity. The number who reported a good perception of understanding of the pharmacologic management of a stable patient with cardiac arrhythmia presimulation increased from 37.7% (60) to 49.1% (78) postsimulation. A Wilcoxon signed-rank test model was fitted to examine improvements in perceptions of understanding of cardiac rhythm identification and the pharmacologic management of cardiac arrhythmia. The results suggested that participation in simulation activities elicited a statistically significant improvement in the students' perceptions of understanding of cardiac rhythm identification and the pharmacologic management of cardiac arrhythmia (p=0.000). CONCLUSIONS: As medical education continues to evolve, simulation-based education may be helpful in enhancing medical students' understanding of cardiac rhythm identification and the pharmacology skills necessary to manage a stable patient with cardiac arrhythmia.

Differential neutralization and inhibition of SARS-CoV-2 variants by antibodies elicited by COVID-19 mRNA vaccines.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence of new variant lineages that have exacerbated the COVID-19 pandemic. Some of those variants were designated as variants of concern/interest (VOC/VOI) by national or international authorities based on many factors including their potential impact on vaccine-mediated protection from disease. To ascertain and rank the risk of VOCs and VOIs, we analyze the ability of 14 variants (614G, Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Eta, Theta, Iota, Kappa, Lambda, Mu, and Omicron) to escape from mRNA vaccine-induced antibodies. The variants show differential reductions in neutralization and replication by post-vaccination sera. Although the Omicron variant (BA.1, BA.1.1, and BA.2) shows the most escape from neutralization, sera collected after a third dose of vaccine (booster sera) retain moderate neutralizing activity against that variant. Therefore, vaccination remains an effective strategy during the COVID-19 pandemic.

The carbon monoxide-releasing molecule, CORM-3 (RU(CO)(3) CL(glycinate)), targets respiration and oxidases in Campylobacter jejuni, generating hydrogen peroxide.

Carbon monoxide (CO) is a classical respiratory inhibitor, but CO-releasing molecules (CO-RMs) have therapeutic value, increasing phagocytosis, and reducing sepsis-induced lethality. CORM-3, Ru(CO)(3) Cl(glycinate), a ruthenium-based carbonyl that liberates CO under physiological conditions, has previously been shown to inhibit bacterial growth and respiration, even at high concentrations of oxygen. Here, we report the effects of CORM-3 on the microaerophilic foodborne pathogen Campylobacter jejuni. Even at CO-RM (i.e., CO) concentrations that exceed dissolved oxygen levels, CORM-3 does not inhibit microaerobic growth. This insensitivity is not due to failure of CORM-3 to penetrate cells, as revealed by assay with extracellular myoglobin and by the ability of CO from externally added CORM-3 to bind intracellular membrane-associated respiratory oxidases. Even at almost 200 µ M oxygen, CORM-3 inhibits formate-dependent respiration and leads to generation of hydrogen peroxide. This work shows that CO-RMs have valuable properties as antimicrobial agents; however, growth inhibition does not always accompany inhibition of respiration, even when ambient oxygen concentrations are low.

CO liberated from a carbon monoxide-releasing molecule exerts a positive inotropic effect in doxorubicin-induced cardiomyopathy.

Carbon monoxide (CO) liberated by a water-soluble carbon monoxide-releasing molecule (CORM-3) induces a positive inotropic effect with a negative chronotropic effect in normal rat hearts. However, the efficacy of CORM-3 under conditions of chronic cardiac insufficiency is unknown. In a rat model of doxorubicin-induced cardiomyopathy, CORM-3 (20 microg/min) produced a positive inotropic effect as demonstrated by significant increases in systolic pressure (P < 0.05) and pressure derivative (dp/dt max) over time (P < 0.05). A similar dose of CO-depleted negative control (inactive CORM-3) failed to cause any change in these parameters. When the inotrope dobutamine was added at a dose of 10 microM following CORM-3, there was no additional increase in systolic pressure or dp/dt max. However, significant rises in systolic pressure and dp/dt max were observed after dobutamine administration to the hearts previously treated with inactive CORM-3. These results suggest that CORM-3 produces a positive inotropic effect in doxorubicin cardiomyopathy rat hearts, similar to that reported previously in normal hearts. The inotropic effect produced by CO in the doxorubicin cardiomyopathy heart was mimicked by a classical inotrope (dobutamine), suggesting that either a maximal inotropic effect is achieved at this dose of CORM-3 or both drugs utilize shared signaling pathways in cardiac muscle.

In vitro and in vivo effects of the carbon monoxide-releasing molecule, CORM-3, in the xenogeneic pig-to-primate context.

BACKGROUND: Carbon monoxide (CO) interferes with inflammatory and apoptotic processes associated with ischemia-reperfusion injury and graft rejection. Here, the in vitro effects of carbon monoxide releasing molecule-3 (CORM-3), a novel water-soluble carbonyl CO carrier, have been investigated on porcine aortic endothelial cells (PAEC) and primate peripheral blood mononuclear cells (PBMC). Furthermore, the pharmacodynamics and pharmacotolerance of CORM-3 after administration of single and multiple doses in the primate have been assessed in view of its potential application in pig-to-primate xenotransplantation models. METHODS: For in vitro studies, PAEC and primate PBMC were exposed for 24, 48 and 72 h to CORM-3 (20 to 1000 microm) and viability was measured using an MTS assay. PAEC and primate PBMC proliferation after exposure to CORM-3 was assessed by CFSE labelling. Proliferation of primate PBMC against irradiated pig lymphocytes was also assessed. Tumor necrosis factor alpha (TNF-alpha) production and Caspase-3 and -7 activity in Concanavalin A (conA)-stimulated primate PBMC were measured following treatment with CORM-3. In vivo, CORM-3 was administered i.v. to cynomolgus monkeys at 4 mg/kg, as single or multiple doses for up to 30 days. The effect of CORM-3 was evaluated by the assessment of production of TNF-alpha and interleukin 1beta following PBMC stimulation with LPS by species-specific ELISA. Complete hematologic and biochemical analyses were routinely performed in treated primates. RESULTS: At concentrations <500 microm, CORM-3 did not alter the viability of PAEC or primate PBMC cultures in vitro, nor did it induce significant levels of apoptosis or necrosis. Interestingly, at concentrations of 300 and 500 microm, significant PAEC proliferation was observed, whilst concentrations > or =50 microm inhibited conA-activated primate lymphocyte proliferation (IC(50) of 345.8 +/- 51.9 microm) and the primate xenogeneic response against pig PBMC. Such responses were demonstrated to be CO-dependent. In addition, CORM-3 significantly inhibited caspase-3 and -7 activity at concentrations between 200 and 500 microm and caused a significant reduction in TNF-alpha production (IC(50) 332.8 +/- 33.9 microm). In vivo, following the administration of multiple doses, TNF-alpha production was significantly reduced in comparison to pre-treatment responses, with decreased levels maintained throughout the study. Moreover, a slight and transient increase in transaminases and bilirubin was observed in animals exposed to multiple doses of CORM-3. CONCLUSIONS: These studies suggest that CORM-3 has anti-inflammatory and immunomodulatory properties in primates that may result in clinical benefit to allo- and xenografted organs.

Objective Assessment of the Early Stages of the Learning Curve for the Senhance Surgical Robotic System.

OBJECTIVE: The purpose of this research is to study the early stages of the Senhance learning curve to report how force feedback impacts learning rate. This serves as an exploratory investigation into assumptions that fellows and faculty will adjust faster to the Senhance in comparison with residents, and that force feedback will not hinder skill acquisition. DESIGN: In this study, participants completed the peg transfer and precision cutting task from the Fundamentals of Laparoscopic Surgery (FLS) manual skills assessment five times each using the Senhance while instrument motion was tracked. SETTING: This study took place in the Surgical Education and Activities Laboratory at Duke University Medical Center. PARTICIPANTS: Participants for this study were residents, fellows, and faculty from Duke University Medical Center in general surgery and gynecology specialties (N = 16). RESULTS: Postulated linear mixed effects models with participant level random effects showed significant improvement with additional attempts for the peg transfer task after adjusting for surgical experience and force feedback respectively for the primary FLS score metric. The secondary metric of total instrument path length also showed improvement (significant decreases) in path length with additional attempts after respectively adjusting for surgical experience and force feedback. CONCLUSIONS: This study investigates the early stages of the learning curve of the Senhance. Exploratory modeling indicates that residents, fellows, and faculty surgeons rapidly adapt to the controls of the Senhance regardless of experience level and force feedback engagement. The results from this study may serve as motivation for future prospective studies that achieve sufficient statistical power with a larger sample size and strict experimental design.