Search

The antiplatelet effect of polyunsaturated fatty acids is primarily attributed to its metabolism to bioactive metabolites by oxygenases, such as lipoxygenases (LOX). Platelets have demonstrated the ability to generate 15-LOX-derived metabolites (15-oxylipins); however, whether 15-LOX is in the platelet or is required for the formation of 15-oxylipins remains unclear. This study seeks to elucidate whether 15-LOX is required for the formation of 15-oxylipins in the platelet and determine their mechanistic effects on platelet reactivity. In this study, 15-HETrE, 15-HETE, and 15-HEPE attenuated collagen-induced platelet aggregation, and 15-HETrE inhibited platelet aggregation induced by different agonists. The observed anti-aggregatory effect was due to the inhibition of intracellular signaling including αIIbβ3 and protein kinase C activities, calcium mobilization, and granule secretion. While 15-HETrE inhibited platelets partially through activation of peroxisome proliferator-activated receptor β (PPARβ), 15-HETE also inhibited platelets partially through activation of PPARα. 15-HETrE, 15-HETE, or 15-HEPE inhibited 12-LOX in vitro, with arachidonic acid as the substrate. Additionally, a 15-oxylipin-dependent attenuation of 12-HETE level was observed in platelets following ex vivo treatment with 15-HETrE, 15-HETE, or 15-HEPE. Platelets treated with DGLA formed 15-HETrE and collagen-induced platelet aggregation was attenuated only in the presence of ML355 or aspirin, but not in the presence of 15-LOX-1 or 15-LOX-2 inhibitors. Expression of 15-LOX-1, but not 15-LOX-2, was decreased in leukocyte-depleted platelets compared to non-depleted platelets. Taken together, these findings suggest that 15-oxylipins regulate platelet reactivity; however, platelet expression of 15-LOX-1 is low, suggesting that 15-oxylipins may be formed in the platelet through a 15-LOX-independent pathway.

Civic education in undergraduate institutions is of vital importance to the civic health of society, but faculty often find it difficult to incorporate civic components into existing courses and lack the resources to incorporate civic outreach into their curriculum. As a result, research has shown that time spend on civic engagement is limited. Additionally, civic engagement is rarely discussed, much less encouraged, outside of political science or other social science classes. In this article, we assess the outcomes of a unique co-curricular civic engagement research and learning program in which undergraduate students are required to work with an external partner, usually a municipal agency or nonprofit, to complete an independent research project. Using quantitative and qualitative student evaluations, we find that students had an overwhelmingly positive experience with the program. They especially appreciated the opportunity to work on "real world" issues in an interdisciplinary setting. They also expressed a desire for a longer program that extended beyond a single semester. We discuss the potential implications of these findings.

Human 15-lipoxygenases (LOX) are critical enzymes in the inflammatory process, producing various pro-resolution molecules, such as lipoxins and resolvins, but the exact role each of the two 15-LOXs in these biosynthetic pathways remains elusive. Previously, it was observed that h15-LOX-1 reacted with 5S-HETE in a non-canonical manner, producing primarily the 5S,12S-diHETE product. To determine the active site constraints of h15-LOX-1 in achieving this reactivity, amino acids involved in the fatty acid binding were investigated. It was observed that R402L did not have a large effect on 5S-HETE catalysis, but F414 appeared to π-π stack with 5S-HETE, as seen with AA binding, indicating an aromatic interaction between a double bond of 5S-HETE and F414. Decreasing the size of F352 and I417 shifted oxygenation of 5S-HETE to C12, while increasing the size of these residues reversed the positional specificity of 5S-HETE to C15. Mutants at these locations demonstrated a similar effect with 7S-HDHA as the substrate, indicating that the depth of the active site regulates product specificity for both substrates. Together, these data indicate that of the three regions proposed to control positional specificity, π-π stacking and active site cavity depth are the primary determinants of positional specificity with 5S-HETE and h15-LOX-1. Finally, the altered reactivity of h15-LOX-1 was also observed with 5S-HEPE, producing 5S,12S-diHEPE instead of 5S,15S-diHEPE (aka resolvin E4 (RvE4). However, h15-LOX-2 efficiently produces 5S,15S-diHEPE from 5S-HEPE. This result is important with respect to the biosynthesis of the RvE4 since it obscures which LOX isozyme is involved in its biosynthesis. Future work detailing the expression levels of the lipoxygenase isoforms in immune cells and selective inhibition during the inflammatory response will be required for a comprehensive understanding of RvE4 biosynthesis.

New results are presented from wind tunnel studies performed at the United States Environmental Protection Agency (U.S. EPA), which include cases with solid roadside barriers of varying heights and cases with varying distances between the line source (roadway) and a 6-m-tall barrier. The Source-to-Barrier Distance cases include seven lanes of traffic with each lane acting as an independent source of continuous emissions along a line (i.e., line source). A mixed-wake algorithm that accounts for barrier effects within a steady-state air dispersion model was updated based on the recent wind tunnel studies. To study the effects of a solid roadside barrier, varying barrier heights and varying distances between the line source and barrier were modeled with the U.S. EPA regulatory air dispersion model AERMOD (v. 21112) using the line-source option that includes an experimental barrier option (RLINEXT). The mixed-wake algorithm reproduced the shape of the vertical concentration profiles observed in the wind tunnel data, including the uniform concentration profile from the ground vertically to a height somewhat greater than the height of the barrier. The algorithm responded appropriately to changes in barrier height and source-to-barrier distance, producing greater reductions in ground-level concentrations for taller barriers and for shorter source-to-barrier distances. Additionally, a rule of thumb that approximates the effect of a downwind barrier was formulated by converting an estimated vertical dispersion into an additional travel distance. The wind tunnel results, the update to the mixed-wake algorithm, and a comparison of the two data sets are described in this paper.

Human epithelial 15-lipoxygenase-2 (h15-LOX-2, ALOX15B) is expressed in many tissues and has been implicated in atherosclerosis, cystic fibrosis and ferroptosis. However, there are few reported potent/selective inhibitors that are active ex vivo. In the current work, we report newly discovered molecules that are more potent and structurally distinct from our previous inhibitors, MLS000545091 and MLS000536924 (Jameson et al, PLoS One, 2014, 9, e104094), in that they contain a central imidazole ring, which is substituted at the 1-position with a phenyl moiety and with a benzylthio moiety at the 2-position. The initial three molecules were mixed-type, non-reductive inhibitors, with IC50 values of 0.34 ± 0.05 μM for MLS000327069, 0.53 ± 0.04 μM for MLS000327186 and 0.87 ± 0.06 μM for MLS000327206 and greater than 50-fold selectivity versus h5-LOX, h12-LOX, h15-LOX-1, COX-1 and COX-2. A small set of focused analogs was synthesized to demonstrate the validity of the hits. In addition, a binding model was developed for the three imidazole inhibitors based on computational docking and a co-structure of h15-LOX-2 with MLS000536924. Hydrogen/deuterium exchange (HDX) results indicate a similar binding mode between MLS000536924 and MLS000327069, however, the latter restricts protein motion of helix-α2 more, consistent with its greater potency. Given these results, we designed, docked, and synthesized novel inhibitors of the imidazole scaffold and confirmed our binding mode hypothesis. Importantly, four of the five inhibitors mentioned above are active in an h15-LOX-2/HEK293 cell assay and thus they could be important tool compounds in gaining a better understanding of h15-LOX-2's role in human biology. As such, a suite of similar pharmacophores that target h15-LOX-2 both in vitro and ex vivo are presented in the hope of developing them as therapeutic agents.

This paper presents an analysis of data from a wind tunnel study conducted to examine the dispersion of emissions at the edges of near-road noise barriers. The study is motivated by the concern that a barrier positioned downwind of a roadway may guide highly polluted plumes along the barrier leading to heightened concentrations as the plume spills around and downwind of the barrier end. The wind tunnel database consists of measurements of dispersion around a simulated roadway segment with various noise barrier configurations. Each roadway segment simulated in the wind tunnel had full-scale equivalent dimensions of 135 m long. Barrier segments, 135 m long with a height (H) of 6 m, were located on the downwind side of the source at a distance of 18 m from it (measured perpendicularly from the line source). Examination of the concentration patterns associated with the cases indicates that 1) vertical mixing induced by barriers persists at crosswind distances up to the edge (lateral end) of the barrier and downwind distances of x/H = 10, 2) concentration levels at all heights below z/H = 1 increase towards the edge of the barrier at downwind distances less than x/H = 7, and 3) concentration is well mixed in the vertical at the edge of the barrier, and the levels can be higher than in the middle of the barrier even when the source ends at the edge of the barrier. We have formulated a parameterization that captures the major features of these observations and can be incorporated in models such as RLINE.

Introduction: The coronavirus 2019 (COVID-19) pandemic has reinforced the importance of facial protection against droplet transmission of diseases. Healthcare workers wear personal protection equipment (PPE), including face shields and masks. Plastic face shields may have advantages over regular medical masks. Although many designs of face shields exist, there is a paucity of evidence regarding the efficacy of shield designs against droplet transmissions. There is even less published evidence comparing various face shields. Due to the urgency of the pandemic and the health and safety of healthcare workers, we aimed to study the efficacy of various face shields against droplet transmission. Methods: We simulated droplet transmission via coughing using a heavy-duty chemical spray bottle filled with fluorescein. A standard-adult sized mannequin head was used. The mannequin head wore various face shields and was positioned to face the spray bottle at either a 0°, 45°, or 90° angle. The spray bottle was positioned at and sprayed from 30 centimeters (cm), 60 cm, or 90 cm away from the head. These steps were repeated for all face shields used. Control was a mannequin that wore no PPE. A basic mask was also tested. We collected data for particle count, total area of particle distribution, average particle size, and percentage area covered by particles. We analyzed percent covered by particles using a repeated measures mixed-model regression with Tukey-Kramer pairwise comparison. Results: We used least square means to estimate the percentage area covered by particles. Wearing PPE regardless of the design reduced particle transmission to the mannequin compared to the control. The LCG mask had the lowest square means of 0.06 of all face-shield designs analyzed. Tukey-Kramer pairwise comparison showed that all PPEs had a decrease in particle contamination compared to the control. LCG shield was found to have the least contamination compared to all other masks (P < 0.05). Conclusion: Results suggest the importance of wearing a protective covering against droplet transmission. The LCG shield was found to decrease facial contamination by droplets the most of any tested protective equipment.

The oxylipins, 5S,12S-dihydroxy-6E,8Z,10E,14Z-eicosatetraenoic acid (5S,12S-diHETE) and 5S,15S-dihydroxy-6E,8Z,11Z,13E-eicosatetraenoic acid (5S,15S-diHETE), have been identified in cell exudates and have chemotactic activity toward eosinophils and neutrophils. Their biosynthesis has been proposed to occur by sequential oxidations of arachidonic acid (AA) by lipoxygenase enzymes, specifically through oxidation of AA by h5-LOX followed by h12-LOX, h15-LOX-1, or h15-LOX-2. In this work, h15-LOX-1 demonstrates altered positional specificity when reacting with 5S-HETE, producing 90% 5S,12S-diHETE, instead of 5S,15S-diHETE, with kinetics 5-fold greater than that of h12-LOX. This is consistent with previous work in which h15-LOX-1 reacts with 7S-HDHA, producing the noncanonical, DHA-derived, specialized pro-resolving mediator, 7S,14S-diHDHA. It is also determined that oxygenation of 5S-HETE by h15-LOX-2 produces 5S,15S-diHETE and its biosynthetic kcat/KM flux is 2-fold greater than that of h15-LOX-1, suggesting that h15-LOX-2 may have a greater role in lipoxin biosynthesis than previously thought. In addition, it is shown that oxygenation of 12S-HETE and 15S-HETE by h5-LOX is kinetically slow, suggesting that the first step in the in vitro biosynthesis of both 5S,12S-diHETE and 5S,15S-diHETE is the production of 5S-HETE.

The two oxylipins 7S,14S-dihydroxydocosahexaenoic acid (diHDHA) and 7S,17S-diHDHA [resolvin D5 (RvD5)] have been found in macrophages and infectious inflammatory exudates and are believed to function as specialized pro-resolving mediators (SPMs). Their biosynthesis is thought to proceed through sequential oxidations of DHA by lipoxygenase (LOX) enzymes, specifically, by human 5-LOX (h5-LOX) first to 7(S)-hydroxy-4Z,8E,10Z,13Z,16Z,19Z-DHA (7S-HDHA), followed by human platelet 12-LOX (h12-LOX) to form 7(S),14(S)-dihydroxy-4Z,8E,10Z,12E,16Z,19Z-DHA (7S,14S-diHDHA) or human reticulocyte 15-LOX-1 (h15-LOX-1) to form RvD5. In this work, we determined that oxidation of 7(S)-hydroperoxy-4Z,8E,10Z,13Z,16Z,19Z-DHA to 7S,14S-diHDHA is performed with similar kinetics by either h12-LOX or h15-LOX-1. The oxidation at C14 of DHA by h12-LOX was expected, but the noncanonical reaction of h15-LOX-1 to make over 80% 7S,14S-diHDHA was larger than expected. Results of computer modeling suggested that the alcohol on C7 of 7S-HDHA hydrogen bonds with the backbone carbonyl of Ile399, forcing the hydrogen abstraction from C12 to oxygenate on C14 but not C17. This result raised questions regarding the synthesis of RvD5. Strikingly, we found that h15-LOX-2 oxygenates 7S-HDHA almost exclusively at C17, forming RvD5 with faster kinetics than does h15-LOX-1. The presence of h15-LOX-2 in neutrophils and macrophages suggests that it may have a greater role in biosynthesizing SPMs than previously thought. We also determined that the reactions of h5-LOX with 14(S)-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-DHA and 17(S)-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-DHA are kinetically slow compared with DHA, suggesting that these reactions may be minor biosynthetic routes in vivo. Additionally, we show that 7S,14S-diHDHA and RvD5 have anti-aggregation properties with platelets at low micromolar potencies, which could directly regulate clot resolution.

This paper focuses on the impact of solid barriers located upwind of a highway in reducing vehicle related concentrations that occur downwind of the roadway, compared to a highway without barriers. Measurements made in the United States Environmental Protection Agency's meteorological wind tunnel show that the mitigating impact of an upwind barrier is comparable to that of a downwind barrier. Upwind barriers lead to reductions in pollution concentrations by drawing emissions in from the highway toward the barrier. The emissions are then entrained into the flow above the recirculation zone and dispersed vertically as they are advected downwind. This upwind transport of vehicle emissions leads to concentrations at the center of the roadways that are roughly 200–300% higher than those measured on roadways with downwind barriers. This difference between on-road concentrations indicates that although both types of barriers mitigate the impact of vehicle emissions downwind of a roadway, the upwind barrier may create adverse air quality impacts for the people on the road. We have formulated a semiempirical dispersion model that incorporates the physics revealed by the wind tunnel measurements. This model improves upon a model proposed by Ahangar et al. (2017) by adjusting the wind speed to get a more realistic plume dispersion just downwind of the upwind barrier and also by providing vertical profiles of concentrations in addition to ground-level concentrations. The upwind barrier model proposed in this paper and the downwind barrier model described in Francisco et al. (2022) have been incorporated into AERMOD (version 21112) as a nonregulatory option, including the new two-barrier option when modeling both barriers on the same roadway. Implications: Our paper presents an air dispersion model algorithm for modeling the effect of upwind noise barriers on dispersion of traffic-related emissions from roadways, which was incorporated into EPA's AERMOD and then evaluated using observations from a wind tunnel experiment. The results are compared and contrasted with results from both a no-barrier case and downwind barrier cases. This manuscript expands on previously published work analyzing the effect of barrier height and source-to-barrier distance on downwind dispersion (Atmos. Pollut. Res., 13:101385, 2022, ). The current manuscript uses the same wind tunnel setup as reported there, but focuses on a different subset of cases, namely the upwind barrier cases, when developing dispersion model algorithms to simulate the observed effects. We believe the evaluations of the vertical profiles from the wind tunnel study, development, and incorporation of the upwind barrier algorithms into AERMOD, and model evaluation of these new algorithms are significant contributions to understanding the effects of these commonly used roadside barriers [ABSTRACT FROM AUTHOR]

Lipoxygenases, important enzymes in inflammation, can regulate their substrate specificity by allosteric interactions with their own hydroperoxide products. In this work, addition of both 13-(S)-hydroxy-(9Z,11E)-octadecadienoic acid [13-(S)-HODE] and 13-(S)-hydroperoxy-(6Z,9Z,11E)-octadecatrienoic acid to human epithelial 15-lipoxygenase-2 (15-LOX-2) increases the kcat/KM substrate specificity ratio of arachidonic acid (AA) and γ-linolenic acid (GLA) by 4-fold. 13-(S)-HODE achieves this change by activating kcat/KM(AA) but inhibiting kcat/KM(GLA), which indicates that the allosteric structural changes at the active site discriminate between the length and unsaturation differences of AA and GLA to achieve opposite kinetic effects. The substrate specificity ratio is further increased, 11-fold in total, with an increase in pH, suggesting mechanistic differences between the pH and allosteric effects. Interestingly, the loss of the PLAT domain affects substrate specificity but does not eliminate the allosteric properties of 15-LOX-2, indicating that the allosteric site is located in the catalytic domain. However, the removal of the PLAT domain does change the magnitude of the allosteric effect. These data suggest that the PLAT domain moderates the communication pathway between the allosteric and catalytic sites, thus affecting substrate specificity. These results are discussed in the context of protein dimerization and other structural changes.

We report the discovery of a novel dual inhibitor targeting fungal sterol 14α-demethylase (CYP51 or Erg11) and human 5-lipoxygenase (5-LOX) with improved potency against 5-LOX due to its reduction of the iron center by its phenylenediamine core. A series of potent 5-LOX inhibitors containing a phenylenediamine core, were synthesized that exhibit nanomolar potency and >30-fold selectivity against the LOX paralogs, platelet-type 12-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity against ovine cyclooxygenase-1 and human cyclooxygnease-2. The phenylenediamine core was then translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound, ketaminazole. Ketaminazole was found to be a potent dual inhibitor against human 5-LOX (IC50 = 700 nM) and CYP51 (IC50 = 43 nM) in vitro. It was tested in whole blood and found to down-regulate LTB4 synthesis, displaying 45% inhibition at 10 µM. In addition, ketaminazole selectively inhibited yeast CYP51 relative to human CYP51 by 17-fold, which is greater selectivity than that of ketoconazole and could confer a therapeutic advantage. This novel dual anti-fungal/anti-inflammatory inhibitor could potentially have therapeutic uses against fungal infections that have an anti-inflammatory component.

We report the discovery of novel small molecule inhibitors of platelet-type 12-human lipoxygenase, which display nanomolar activity against the purified enzyme, using a quantitative high-throughput screen (qHTS) on a library of 153607 compounds. These compounds also exhibit excellent specificity, >50-fold selectivity vs the paralogues, 5-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity vs ovine cyclooxygenase-1 and human cyclooxygenase-2. Kinetic experiments indicate this chemotype is a noncompetitive inhibitor that does not reduce the active site iron. Moreover, chiral HPLC separation of two of the racemic lead molecules revealed a strong preference for the (-)-enantiomers (IC(50) of 0.43 ± 0.04 and 0.38 ± 0.05 μM) compared to the (+)-enantiomers (IC(50) of >25 μM for both), indicating a fine degree of selectivity in the active site due to chiral geometry. In addition, these compounds demonstrate efficacy in cellular models, which underscores their relevance to disease modification.