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The role of cuticle changes in insecticide resistance in the major malaria vector Anopheles gambiae was assessed. The rate of internalization of 14C deltamethrin was significantly slower in a resistant strain than in a susceptible strain. Topical application of an acetone insecticide formulation to circumvent lipid-based uptake barriers decreased the resistance ratio by ∼50%. Cuticle analysis by electron microscopy and characterization of lipid extracts indicated that resistant mosquitoes had a thicker epicuticular layer and a significant increase in cuticular hydrocarbon (CHC) content (∼29%). However, the CHC profile and relative distribution were similar in resistant and susceptible insects. The cellular localization and in vitro activity of two P450 enzymes, CYP4G16 and CYP4G17, whose genes are frequently overexpressed in resistant Anopheles mosquitoes, were analyzed. These enzymes are potential orthologs of the CYP4G1/2 enzymes that catalyze the final step of CHC biosynthesis in Drosophila and Musca domestica, respectively. Immunostaining indicated that both CYP4G16 and CYP4G17 are highly abundant in oenocytes, the insect cell type thought to secrete hydrocarbons. However, an intriguing difference was indicated; CYP4G17 occurs throughout the cell, as expected for a microsomal P450, but CYP4G16 localizes to the periphery of the cell and lies on the cytoplasmic side of the cell membrane, a unique position for a P450 enzyme. CYP4G16 and CYP4G17 were functionally expressed in insect cells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrate and thus has bona fide decarbonylase activity similar to that of dmCYP4G1/2. The data support the hypothesis that the coevolution of multiple mechanisms, including cuticular barriers, has occurred in highly pyrethroid-resistant An. gambiae.

Insect pest management is a challenging problem across certain insect species. Mass attacks by pine bark beetles are difficult to control because beetles spend most of their life under the bark of trees and mass attacks often occur in remote regions. In other species, insecticide resistance has rendered several classes of insecticide obsolete. Development of novel targets is essential for insect management and control across all taxa. In this dissertation, I present results from experiments on specialized enzymes involved in lipid metabolism through two pathways, cuticular hydrocarbon biosynthesis and isoprenoid pheromone biosynthesis, pathways that provide unique targets for insecticides.Cuticular hydrocarbons are essential to insects in order to prevent water loss and also serve in chemical communication through contact pheromones. The final step in cuticular hydrocarbon biosynthesis is catalyzed by an insect specific cytochrome P450 from the CYP4G family. While most insects have two CYP4G genes, not all CYP4G gene products are hydrocarbon forming enzymes while other CYP4G enzymes appear to have dual functions. Here I show evidence that at least one CYP4G gene from each of Musca domestica, Dendroctonus ponderosae, Anopheles gambiae, and Aedes aegypti is involved in cuticular hydrocarbon biosynthesis. Apis mellifera is unique in that it has one CYP4G gene; it functions in hydrocarbon production. D. ponderosae has two CYP4G genes that appear to be involved in both hydrocarbon synthesis and pheromone production via theexo- brevicomin pathway.Chemical communication between insects is not restricted to non-volatile contact pheromones. Some insects rely on volatile chemicals to communicate over relatively long distances. Bark beetles from the Ips species use a combination of ipsdienol and ipsenol to coordinate mass attacks on host trees. The ratio and enantiomeric blends of ipsdienol and ipsenol differ between species and even with populations. Several enzymes in the monoterpenoid pathway have been characterized, however key enzymes in the pathway are yet to be discovered. A central intermediate in all ipsdienol and ipsenol biosynthetic pathways is ipsdienone. While oxidation of ipsdienol and ipsenol forms their respective ketones, it is not known how the carbon pool shifts from ipsdienone (with 3 carbon-carbon double bonds) to ipsenone (2 carbon-carbon double bonds). In this dissertation, I present evidence for a novel monoterpenoid carbon-carbon double bond reductase that catalyzes a central step in the ipsdienol and ipsenol biosynthesis. This enzyme, ipsdienone reductase (IDONER), converts ipsdienone to ipsenone, and is the first monoterpenoid carbon double bond reductase to be biochemically characterized in animals.Data in this dissertation allow for the prediction that at least one CYP4G in each species is involved in hydrocarbon production. In addition, reduction of ipsdienone by IDONER contributes to the final pheromone composition of Ips species. Future work to identify, model, and characterize other specialized enzymes in lipid metabolism will provide a map to understanding how insects have evolved the ability to simultaneously protect themselves and communicate, ultimately aiding in identification of targets for pest management.

Demand for fine chemicals and value-added products has increased annually concomitantly with the rapid expansion of biocatalytic production of these chemicals. With an increased mandate for chemicals in the pharmaceutical, agricultural, scents and flavorants industry, it is essential to identify enzymes capable of performing specific, complex chemical reactions in an efficient, economical and environmentally friendly manner. Insects produce secondary metabolites that are potential value-added chemicals, therefore insect enzymes provide a rich source of catalysts that can be added to the collection of biocatalysts used to perform these intricate tasks. This thesis explores the biochemical role of three insect enzymes, CYP9T3, MPB-CPR, and CG4020, which are involved in lipid metabolism and could potentially be used as biocatalysts for value added products. All three enzymes were expressed with a baculoviral system in Sf9 cells and used in functional assays. Eastern Ips pini CYP9T3 is a cytochrome P450 with 94% sequence identity to western Ips pini CYP9T2. CYP9T3 accepts myrcene, (+)- and (-)-α-pinene, (+)- and (-)- limonene, and (+)-3-carene as substrates, a pattern similar to CYP9T2. However, the enantiomeric ratio of (4R)-(-)-ipsdienol to (4S)-(+)-ipsdienol is significantly different between CYP9T3 and CYP9T2. Additionally, the product from (-)-α-pinene is trans-verbenol with CYP9T3 and myrtenol with CYP9T2. Assays with β-pinene, terpinolene, gamma-terpinene, α-phellandrene did not yield detectable products by either CYP9T3 or CYP9T2. Cytochromes P450 require the transfer of electrons from a protein partner, commonly cytochrome P450 reductases. Mountain pine beetle cytochrome P450 reductase (MPB-CPR) is the first bark beetle cytochrome P450 reductase to be isolated and characterized. MPB-CPR is 69% identical to house fly-CPR (HF-CPR), the reductase used in previous bark beetle P450 functional assays. Recombinant MPB-CPR microsomes reduce cytochrome c with apparent K m and vmax of 85.04 μM and 8.42 nmol˙min-1 ˙μg total protein-1, respectively. Initial kinetic assays with CYP9T3 indicate that MPB-CPR reduces the P450 in the presence of myrcene with apparent Km and vmax values of 15.8 uM and 11.3 nmol˙min-1·U enzyme-1, respectively. The final step in hydrocarbon biosynthesis in insects is the removal of one carbon from a fatty aldehyde through the action of an oxidative decarbonylase, CYP4G2. Experimental evidence from M. domestica microsomal preparations indicates that a fatty acyl-CoA is reduced to a fatty aldehyde prior to the oxidative decarbonylation reaction and the candidate enzyme is a fatty acyl-CoA reductase (FAR). CG4020, a Drosophila melanogaster FAR, has an expression pattern similar to CYP4G1, the D. melanogaster homologue of CYP4G2. Additionally, suppression of CG4020 produces flies with fifty percent less total hydrocarbon than wild type flies. Preliminary assays with expressed CG4020 did not confirm the biochemical function of this enzyme, however they did provide relevant background information from which future experiments can be designed.

The cytochrome P450 enzymes of the CYP4G subfamily are some of the most enigmatic insect P450s. The dipterans with sequenced genomes have two CYP4G paralogs. In , CYP4G1 is highly expressed in the oenocytes and catalyzes the last enzymatic step in the biosynthesis of cuticular hydrocarbons. In contrast, CYP4G15 is expressed in the brain glial cells, but its function is unknown. The genome encodes two CYP4Gs: CYP4G36 (ortholog of DmCYP4G1) and CYP4G35 (ortholog of DmCYP4G15). Here, we show that CYP4G35 is highly expressed in mosquito antennae, and the RNAi knockdown of CYP4G35 results in delayed host-seeking. CYP4G knockout lines confirmed delayed host-seeking behavior in CYP4G35 knockout females. Proteomics analysis of CYP4G35 KO females also corroborates the physiological findings and shows upregulation of proteins related to olfaction and other CYP4Gs to compensate for the lack of CYP4G35. Immunohistochemistry and hybridization were used to localize CYP4G35 and demonstrated its expression in the sensilla lymph of the antennae and the tip of the proboscis. CYP4G35 and CYP4G36 fusion proteins with cytochrome P450 reductase demonstrated that, unlike CYP4G36, CYP4G35 lacks an oxidative decarbonylase function. Together, our data support a novel function of CYP4G35 in modulating olfactory response.

Across mammals, increased body size is positively associated with lifespan. However, within species, this relationship is inverted. This is well illustrated in dogs (Canis familiaris), where larger dogs exhibit accelerated life trajectories: growing faster and dying younger than smaller dogs. Similarly, some age-associated traits (e.g., growth rate and physiological pace of aging) exhibit accelerated trajectories in larger breeds. Yet, it is unknown whether cognitive performance also demonstrates an accelerated life course trajectory in larger dogs. Here, we measured cognitive development and aging in a cross-sectional study of over 4000 dogs from 66 breeds using nine memory and decision-making tasks performed by citizen scientists as part of the Dognition project. Specifically, we tested whether cognitive traits follow a compressed (accelerated) trajectory in larger dogs, or the same trajectory for all breeds, which would result in limited cognitive decline in larger breeds. We found that all breeds, regardless of size or lifespan, tended to follow the same quadratic trajectory of cognitive aging—with a period of cognitive development in early life and decline in later life. Taken together, our results suggest that cognitive performance follows similar age-related trajectories across dog breeds, despite remarkable variation in developmental rates and lifespan.

Plant nucleotide binding, leucine-rich repeat (NLR) receptors detect pathogen effectors and initiate an immune response. Since their discovery, NLRs have been the focus of protein engineering to improve disease resistance. However, this approach has proven challenging, in part due to their narrow response specificity. Previously, we revealed the structural basis of pathogen recognition by the integrated heavy metal associated (HMA) domain of the rice NLR Pikp (Maqbool et al., 2015). Here, we used structure-guided engineering to expand the response profile of Pikp to variants of the rice blast pathogen effector AVR-Pik. A mutation located within an effector-binding interface of the integrated Pikp–HMA domain increased the binding affinity for AVR-Pik variants in vitro and in vivo. This translates to an expanded cell-death response to AVR-Pik variants previously unrecognized by Pikp in planta. The structures of the engineered Pikp–HMA in complex with AVR-Pik variants revealed the mechanism of expanded recognition. These results provide a proof-of-concept that protein engineering can improve the utility of plant NLR receptors where direct interaction between effectors and NLRs is established, particularly where this interaction occurs via integrated domains.

Carbon sequestration in deep underground saline aquifers holds significant promise for reducing atmospheric carbon dioxide emissions (CO2). However, challenges remain in predicting the long term migration of injected CO2. Addressing these challenges requires an understanding of pore-scale transport of CO2 within existing brine-filled geological reservoirs. Studies on the transport of fluids through geological porous media have predominantly focused on oil-bearing formations such as sandstone. However, few studies have considered pore-scale transport within limestone and other carbonate formations, which are found in potential storage sites. In this work, high-resolution micro-Computed Tomography (microCT) was used to obtain pore-scale structural information of two model carbonates: Indiana Limestone and Pink Dolomite. A modified watershed algorithm was applied to extract pore network from the reconstructed microCT volumetric images of rock samples and compile a list of pore-scale characteristics from the extracted networks. These include statistical distributions of pore size and radius, pore-pore separation, throat radius, and network coordination. Finally, invasion percolation algorithms were applied to determine saturation-pressure curves for the rock samples. The statistical distributions were comparable to literature values for the Indiana Limestone. This served as validation for the network extraction approach for Pink Dolomite, which has not been considered previously. Based on the connectivity and the pore-pore separation, formations such as Pink Dolomite may present suitable storage sites for carbon storage. The pore structural distributions and saturation curves obtained in this study can be used to inform core- and reservoir-scale modeling and experimental studies of sequestration feasibility. La séquestration du carbone dans les aquifères salins profonds souterrains est très prometteuse pour la réduction des émissions de dioxyde de carbone (CO2) dans l’atmosphère. Cependant, des problèmes demeurent dans la prédiction de la migration à long terme du CO2 injecté. Relever ces défis nécessite une compréhension du transport de CO2 à l’échelle du pore dans des réservoirs géologiques existants remplis de saumure. Les études sur le transport des fluides en milieu poreux géologique ont principalement porté sur les formations oléagineuses telles que le grès. Cependant, peu d’études ont examiné les transports à l’échelle du pore dans le calcaire et d’autres formations carbonatées, qui se trouvent dans des sites de stockage potentiels. Dans ce travail, la micro-tomographie (microCT) à haute résolution a été utilisée pour obtenir de l’information structurale à l’échelle du pore de deux exemples de formations carbonatées : Indiana limestone et Pink dolomite. Un algorithme watershed a été appliqué pour extraire les réseaux de pores des microCT images volumétriques reconstruits des spécimens de roche et de compiler une liste de caractéristiques pores des réseaux extraits. Il s’agit notamment de distributions statistiques de la taille et du rayon des pores, la séparation entres pores, le rayon des gorges, et la coordination du réseau. Enfin, des invasions de percolation ont été appliquées pour déterminer les courbes de saturation pression pour les exemples de roches. Les distributions statistiques sont comparables aux valeurs de la littérature pour l’Indiana limestone. Cela a servi de validation de l’approche d’extraction de réseau pour Pink dolomite, qui n’a pas été précédemment examinés. Basées sur la connectivité et la séparation entre pores, les formations Aelles que Pink dolomite peuvent présenter des sites de stockage appropriés pour le stockage du CO2. Les distributions de structure des pores et des courbes de saturation obtenus dans cette étude peuvent être utilisées pour informer noyau et modélisation à l’échelle du réservoir et les études expérimentales de la faisabilité de la séquestration.

Skillful, voluntary movements are underpinned by computations performed by networks of interconnected neurons in the primary motor cortex (M1). Computations are reflected by patterns of coactivity between neurons. Using pairwise spike time statistics, coactivity can be summarized as a ( ). Here, we show that the structure of FNs constructed from an instructed-delay reach task in nonhuman primates is behaviorally specific: Low-dimensional embedding and graph alignment scores show that FNs constructed from closer target reach directions are also closer in network space. Using short intervals across a trial, we constructed and found that temporal FNs traverse a low-dimensional subspace in a reach-specific trajectory. Alignment scores show that FNs become separable and correspondingly decodable shortly after the cue. Finally, we observe that reciprocal connections in FNs transiently decrease following the cue, consistent with the hypothesis that information external to the recorded population temporarily alters the structure of the network at this moment. It remains unclear how motor cortical neurons flexibly perform the computations necessary to generate movement. We hypothesized that neuronal coactivity contains movement information, and its dynamics can reveal how the population switches computations during a task. We quantified coactivity as a functional network (FN) with single neurons as nodes and population coactivity as directed weighted edges. We also constructed FNs within short epochs across a trial to determine coactivity begins to carry information and to investigate the dynamic structure of these interactions. Following the cue, reciprocal connections in FNs transiently decrease, and shortly after, FNs become maximally decodable for reach direction.

Background. Preterm Premature Rupture of Membranes (PPROM) is a major leading cause of preterm births. While the cause for PPROM remains unidentified, it is anticipated to be due to subclinical infection, since a large proportion of PPROM patients display signs of chorioamnionitis. Since subclinical infections can be facilitated by dysbiosis, our goal was to characterize the vaginal microbiome and amniotic fluid discharge upon PPROM, through latency antibiotic treatment, and until delivery, to detect the presence of pathogens, microbiota alteration, and microbial response to treatment. Methods. Enrolled subjects (15) underwent routine institutional antenatal care for PPROM, including the administration of latency antibiotics. Serial vaginal swabs were obtained from diagnosis of PPROM through delivery and the sequencing of the V3–V5 region of the 16S rRNA gene was performed for all collected samples. Results. The results show that Lactobacilli species were markedly decreased when compared to vaginal swabs collected from uncomplicated pregnancy subjects with a matched gestational time. Prevotella and Peptoniphilus were the most prevalent taxa in PPROM subjects at presentation. The vaginal microbiome of the PPROM subjects varied substantially intra- and inter-subjects. Several taxa were found to be significantly reduced during and after the antibiotic treatment: Weeksella , Lachnospira , Achromobacter , and Pediococcus . In contrast, Peptostreptococcus and Tissierellaceae ph2 displayed a significant increase after the antibiotic treatment. However, the relative abundance of Lactobacillus , Prevotella , and Peptoniphilus was not substantially impacted during the hospitalization of the PPROM subjects. The deficiency of Lactobacillus , and constancy of known pathogenic species, such as Prevotella and Peptoniphilus during and after antibiotics, highlights the persistent dysbiosis and warrants further investigation into mitigating approaches. Discussion. PPROM is responsible for one third of all preterm births. It is thought that subclinical infection is a crucial factor in the pathophysiology of PPROM because 25–40% of patients present signs of chorioamnionitis on amniocentesis. Here we sought to directly assess the bacterial content of the vagina and leaking amniotic fluid of subjects at presentation, throughout treatment and up until delivery, in order to search for common pathogens, microbiota changes, and microbial response to latency antibiotic treatment. We have found that the vaginal microbiome of PPROM subjects is highly variable and displays significant changes to treatment. However, the unchanging deficiency of Lactobacillus , and persistence of known pathogenic species, such as Prevotella and Peptoniphilus from presentation, through antibiotic treatment and up until delivery, highlights the persistent dysbiosis and warrants further investigation into mitigating approaches.