Studies of G Protein-Coupled Receptor Stability and Dimerization Using Novel Fluorescence and Crosslinking Approaches

G protein-coupled receptors (GPCRs) comprise a large family of related seventransmembrane- helical membrane proteins that bind to specific extracellular ligands, such as hormones or neuromodulators. The active receptor-ligand complex then engages with a heterotrimeric G protein on the cytoplasmic surface of the plasma membrane to facilitate a change in the concentration of an intracellular second messenger, such as cAMP. A number of non-canonical signaling pathways, such as β-arrestin-mediated signaling, also exist for many, if not all, GPCRs. Receptor signaling is attenuated by phosphorylation and receptor internalization. Recent advances in structural studies of GPCRs have revealed high-resolution structures of both inactive and active receptors in complex with various ligands. Endogenous ligands, drugs and the membrane environment, and even oligomerization can affect receptor signaling efficacy, but the mechanistic details underlying these allosteric effects are poorly characterized. To study allosterism of a ligand-receptor complex in a bilayer requires at least partial enrichment or isolation of the basic signaling unit. Many studies have employed biochemical purification and reconstitution strategies, but GPCRs are inherently unstable when extracted from native membranes so conditions must be carefully selected to preserve receptor integrity. To monitor the functional state of GPCRs during purification and reconstitution, a novel homogeneous time-resolved fluorescence resonance energy transfer (FRET)-based analytical assay was developed. To enable the assay, a novel bioconjugation method was invented to prepare microgram quantities of monoclonal antibodies labeled with long-lived lanthanide fluorophores. As a proof-of-concept, the folding and stability of human C-C chemokine receptor 5 (CCR5), the primary coreceptor for HIV-1 cellular entry, was studied. The assay enabled high-throughput detection of femtomole quantities of CCR5. Thermal denaturation measurements demonstrated that small molecule antagonists substantially stabilize CCR5 and also revealed that the ligands induce distinct receptor conformations, consistent with the hypothesis that GPCRs access numerous conformations during signal transduction rather than operating as a binary active-inactive switch. In addition, high-throughput stability screens led directly to the identification of CCR5 mutants that should be sufficiently stable for crystallization and lead to a high-resolution structure of CCR5, which would significantly advance understanding of the structural basis of HIV entry. The FRETbased assay was also applied to devise and optimize a protocol to incorporate CCR5 into an artificial membrane scaffold called nanoscale apolipoprotein bound bilayers (NABBs). CCR5 was shown to retain proper folding in NABBs and proof-of-concept fluorescence correlation spectroscopy (FCS) experiments were carried out to characterize these structures. Novel FCS standard reagents were developed to facilitate these measurements. The biochemical and analytical approaches reported may be adapted to prepare stable, functional samples of other GPCRs for structural and dynamic studies of receptor allostery. GPCRs are known to form dimers and higher-order oligomers, and despite a growing body of evidence that these complexes are functionally important, the structural basis of receptor-receptor interactions remains unknown. To address this problem, a potential dimerization interface of the prototypical GPCR, rhodopsin, was analyzed using a proteomics approach involving chemical crosslinking and liquid chromatography-mass spectrometry. The strategy was devised so that rhodopsin could be probed in the unique native environment of the rod cell disk membrane. Crosslinking results supported a model of rhodopsin dimerization involving contacts in transmembrane helix 1 and an amphipathic cytoplasmic helix at the carboxyl-terminal tail of the receptor. This novel interface is postulated to be relevant for understanding GPCR oligomerization in general

In-field and Greenhouse Assessments of a Selection of Preemergent Herbicides on Newly Planted Blackberries

This field trial assesses some of the preemergence herbicide options available to growers and their effect on newly transplanted blackberries (Rubus L. subgenus Rubus Watson). Weed control has recently been surveyed as a top priority for blackberry growers; however, limited preemergence herbicides are registered for new blackberry plantings. Weed control is an ongoing component of blackberry production and with few in-season postemergence herbicide options available, growers rely on preemergence herbicides to maintain clean fields. The preemergent herbicides assessed in this trial were chosen with the intention to broaden the chemical control options available to growers in new plantings. A two-year field trial was initiated in 2021 and conducted at two locations: Milo J. Shult Research and Extension Center in Fayetteville, AR (36.09 ºN, 94.17 ºW) and the University of Arkansas Fruit Research Station in Clarksville, AR (35.53 ºN, 93.40 ºW). Seven treatments consisting of six preemergence herbicides (mesotrione, flumioxazin, oryzalin, S-metolachlor, pendimethalin, and napropamide) and one hand-weeded check were applied to field plots of newly transplanted tissue culture propagated blackberry plugs (var. ‘Ouachita’). Preemergence herbicide treatments were reapplied to the same plots in 2022. Data were collected on visual injury, plant height, leaf chlorophyll content, and green coverage of blackberry canopies and of bare ground portions of each plot. Yield data were collected in the second year, and fruit were analyzed for soluble solids content (°Brix), pH, and average berry weight. In the first year mesotrione and flumioxazin treatments caused the most injury to the primocanes. Injury by flumioxazin was not detectable at the final rating of the first year, but injury by mesotrione was high 84 days after treatment (DAT). Napropamide, S-metolachlor, oryzalin, and pendimethalin did not cause injury over 6% throughout the 2021 season. In the second year (2022) no damage was incurred by any treatments, from the treated or the non-chemical weed-free (NCWF) check. The mesotrione treatment affected plant height the most in 2021 at the end of the season compared to the NCWF check. In 2022 plant height was not assessed. Yield measurements taken in 2022 exhibited no significant differences in response to preemergence herbicide treatments. A greenhouse experiment was conducted to investigate the effects of a broad selection of preemergence herbicides at two rates. This screening was initiated August 2021 and repeated March 2022 in Fayetteville, AR in a horticultural greenhouse at the Milo J. Shult Research and Extension Center. Tissue cultured ‘Ouachita’ blackberry plugs were transplanted into utility pots that were treated with a preemergence herbicide treatment. Twenty-five treatments in total consisted of twelve preemergence herbicides at 1× and 2× rates and one untreated control. Data were collected on plant height, visual injury ratings, internode length, leaf chlorophyll content, and destructive harvest including leaf count, leaf dry biomass, specific leaf area (SLA), and leaf area to dry matter ratio (LADMR). Halosulfuron, rimsulfuron, and mesotrione treatments showed progressively increasing visual injury from 7 days after treatment (DAT) until 42 DAT. Flumioxazin, napropamide, S-metolachlor, and pendimethalin treatments exhibited similar responses to the untreated control regarding height and visual injury and may be acceptable for use in young blackberry weed management programs. Data obtained from this screening characterized the physiological response of new blackberries treated with these preemergence herbicides. Both trials demonstrated the deleterious effects of mesotrione on young plants and why it is not recommended for use in first year plantings. Both trials demonstrated the validity of the 24(c) labeling of S-metolachlor. These findings validate many of the regional recommendations and provide new evidence to consider expanding registration and labeled usage requirements for select preemergence herbicides. This knowledge and further field investigation have the potential to lead to more informed IPM strategies

Laughing Together: Comedic Theatre as a Mechanism of Survival during the Holocaust

This thesis aims to analyze the ways many Jewish victims of the Holocaust used comedic theatre to help them overcome their dire circumstances by exploring the high demand for comedic performance in both the ghettoes and concentration camps and analyzing the numerous comedic works that were penned amidst the terror and catastrophic loss surrounding the Jews at this time. The second portion delves into the therapeutic values of comedy and explores the ways laughing may have benefitted those who partook in comedic theatre events. The final chapter investigate whether or not laughter should still be used to help cope with the calamitous events of the Holocaust. Many Jewish victims chose to laugh about their circumstances, but should we, those far removed from the actual events, be able to use comedy without facing a dilemma of morals

Pediatric Giant Right Atrial Aneurysm: A Case Series and Review of the Literature

Giant right atrial aneurysm is a rare form of congenital heart disease with a wide spectrum of clinical presentation varying from asymptomatic patients to those with refractory atrial arrhythmias or severe airway obstruction. Diagnosis is often confused with other causes of right atrial dilation such as E bstein disease. Because of its rare occurrence and variable clinical presentation, inconsistencies in medical and surgical management strategies exist between centers. We present five cases of giant right atrial aneurysm managed at our institution and discuss the clinical presentation, diagnostic challenges, and medical and surgical management.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/107528/1/chd12079.pd

Factors Influencing the Need for and Access to IVF Treatment

Infertility is defined as the inability for a couple to become pregnant after 12 months of regular unprotected sexual intercourse. Infertility can stem from an issue with the female reproductive tract, the male reproductive tract, or both. Individuals struggling with infertility seek medical assistance for a successful reproductive course. However, there are many aspects outside of pathology that may encourage or deter an individual to elect for medical assistance such as in vitro fertilization (IVF). In vitro fertilization is defined as a medical procedure in which an egg is fertilized outside the body. The increased usage of IVF demonstrates the need for equitable access to IVF care. The purpose of this literature review is to consider all the factors and challenges involved in one’s decision to utilize IVF

Quantifying SAGE II (1984–2005) and SAGE III/ISS (2017–2022) observations of smoke in the stratosphere

Using a common analysis approach for data sets produced by the Stratospheric Aerosol and Gas Experiment instruments SAGE II and SAGE III/ISS, we identify 13 likely smoke events based on enhancements in the aerosol extinction coefficient. Nine of these are sufficiently large compared to ambient aerosol levels to compute mean mid-latitude 1020 nm optical depth enhancements that range from 0.0005 to 0.011. We also note that, for large events, the 525 to 1020 nm aerosol extinction coefficient ratio asymptotes at a high extinction coefficient to values between 2 and 3, suggesting that the aerosol radius is relatively small (&lt; 0.3 µm) and relatively consistent from event to event. Most of these events are primarily confined to the lower stratosphere and rarely can be observed above 20 km. We also infer an increase in the frequency of smoke events between the SAGE II (1984–1991, 1996–2005) and SAGE III/ISS (2017-present) periods by almost a factor of 2 and also note that the two largest events occur in the latter data set. However, given the low frequencies overall, we are not confident that the differences can be attributed to changes between the two periods. We also attempt to disentangle the mixing of aerosol in the Northern Hemisphere summer of 1991 from a pyrocumulus event (Baie-Comeau, Quebec) and Mt. Pinatubo and conclude that, while there is evidence for smoke in the lower stratosphere, virtually all of the enhanced aerosol observations in the northern mid-latitudes in the summer of 1991 are associated with the Mt. Pinatubo eruption.</p

Gas phase acetic acid and its qualitative effects on snow crystal morphology and the quasi-liquid layer

A chamber was constructed within which snow crystals were grown on a string at various temperatures, relative humidities, and acetic acid gas phase mole fractions. The temperature, relative humidity, and acid mole fraction were measured for the first time at the point of crystal growth. Snow crystal morphological transition temperature shifts were recorded as a function of acid mole fraction, and interpreted according to the calculated acid concentration in the crystal's quasi-liquid layer, which is believed to have increased in thickness as a function of acid mole fraction, thereby affecting the crystal's morphology consistent with the hypothesis of Kuroda and Lacmann. Deficiencies in the understanding of the quasi-liquid layer and its role in determining snow crystal morphology are briefly discussed