Signal transduction at GPCRs: Allosteric activation of the ERK MAPK by ß-arrestin.

ß-arrestins are multivalent adaptor proteins that bind active phosphorylated G protein-coupled receptors (GPCRs) to inhibit G protein signaling, mediate receptor internalization, and initiate alternative signaling events. ß-arrestins link agonist-stimulated GPCRs to downstream signaling partners, such as the c-Raf-MEK1-ERK1/2 cascade leading to ERK1/2 activation. ß-arrestins have been thought to transduce signals solely via passive scaffolding by facilitating the assembly of multiprotein signaling complexes. Recently, however, ß-arrestin 1 and 2 were shown to activate two downstream signaling effectors, c-Src and c-Raf, allosterically. Over the last two decades, ERK1/2 have been the most intensely studied signaling proteins scaffolded by ß-arrestins. Here, we demonstrate that ß-arrestins play an active role in allosterically modulating ERK kinase activity in vitro and within intact cells. Specifically, we show that ß-arrestins and their GPCR-mediated active states allosterically enhance ERK2 autophosphorylation and phosphorylation of a downstream ERK2 substrate, and we elucidate the mechanism by which ß-arrestins do so. Furthermore, we find that allosteric stimulation of dually phosphorylated ERK2 by active-state ß-arrestin 2 is more robust than by active-state ß-arrestin 1, highlighting differential capacities of ß-arrestin isoforms to regulate effector signaling pathways downstream of GPCRs. In summary, our study provides strong evidence for a new paradigm in which ß-arrestins function as active "catalytic" scaffolds to allosterically unlock the enzymatic activity of signaling components downstream of GPCR activation.

Symbiosis preservation: Putative regulation of fatty acyl-CoA reductase by miR-31a within the symbiont harboring bacteriome through tsetse evolution.

Tsetse flies are the sole vectors of African trypanosomes. In addition to trypanosomes, tsetse harbor obligate Wigglesworthia glossinidia bacteria that are essential to tsetse biology. The absence of Wigglesworthia results in fly sterility, thus offering promise for population control strategies. Here, microRNA (miRNAs) and mRNA expression are characterized and compared between the exclusive Wigglesworthia-containing bacteriome and adjacent aposymbiotic tissue in females of two evolutionarily distant tsetse species (Glossina brevipalpis and G. morsitans). A total of 193 miRNAs were expressed in either species, with 188 of these expressed in both species, 166 of these were novel to Glossinidae, and 41 miRNAs exhibited comparable expression levels between species. Within bacteriomes, 83 homologous mRNAs demonstrated differential expression between G. morsitans aposymbiotic and bacteriome tissues, with 21 of these having conserved interspecific expression. A large proportion of these differentially expressed genes are involved in amino acid metabolism and transport, symbolizing the essential nutritional role of the symbiosis. Further bioinformatic analyses identified a sole conserved miRNA::mRNA interaction (miR-31a::fatty acyl-CoA reductase) within bacteriomes likely catalyzing the reduction of fatty acids to alcohols which comprise components of esters and lipids involved in structural maintenance. The Glossina fatty acyl-CoA reductase gene family is characterized here through phylogenetic analyses to further understand its evolutionary diversification and the functional roles of members. Further research to characterize the nature of the miR-31a::fatty acyl-CoA reductase interaction may find novel contributions to the symbiosis to be exploited for vector control.

The Association Between Korean American Nurse and Primary Care Provider Burnout, Areas of Worklife, and Perceptions of Pandemic Experience: Cross-sectional Study.

BACKGROUND: Korean immigrants are among the fastest-growing ethnic minority groups and make up the fifth-largest Asian group in the United States. A better understanding of the work environment factors and its impact on Korean American nurse and primary care provider (PCP) burnout may guide the development of targeted strategies to help mitigate burnout and workplace stressors, which is critical for the retention of Korean American nurses and PCPs to promote better alignment of national demographic trends and meet patients' preference for cultural congruence with their health care providers (HCPs). Although there is a growing number of studies on HCP burnout, a limited number of studies specifically focus on the experience of ethnic minority HCPs, particularly during the COVID-19 pandemic. OBJECTIVE: In light of these gaps in literature, the aim of this study was to assess burnout among Korean American HCPs and to identify work conditions during a pandemic that may be associated with Korean American nurse and PCP burnout. METHODS: A total of 184 Korean American HCPs (registered nurses [RNs]: n=97; PCPs: n=87) practicing in Southern California responded to a web-based survey between February and April 2021. The Maslach Burnout Inventory, Areas of Worklife Survey, and Pandemic Experience & Perceptions Survey were used to measure burnout and work environment factors during the pandemic. A multivariate linear regression analysis was used to assess work environment factors associated with the 3 subcategories of burnout. RESULTS: No significant differences were found in the level of burnout experienced by Korean American nurses and PCPs. For RNs, greater workload (P<.001), lower resource availability (P=.04), and higher risk perception (P=.02) were associated with higher emotional exhaustion. Greater workload was also associated with higher depersonalization (P=.003), whereas a greater (professional) community (P=.03) and higher risk perception (P=.006) were associated with higher personal accomplishment. For PCPs, greater workload and poor work-life balance were associated with higher emotional exhaustion (workload: P<.001; worklife: P=.005) and depersonalization (workload: P=.01; worklife: P<.001), whereas only reward was associated with personal accomplishment (P=.006). CONCLUSIONS: Findings from this study underscore the importance of strategies to promote a healthy work environment across multiple levels that recognize demographic variation among Korean American RNs and PCPs, potentially influencing their burnout mitigation needs. A growing recognition of identity-informed burnout experiences across frontline Korean American RNs and PCPs argues for future explorations that capture nuance both across and within this and other ethnic minority nurse and PCP groups. By recognizing and capturing these variations, we may better support the creation of targeted, burnout-mitigating strategies for all.

The Tsetse Metabolic Gambit: Living on Blood by Relying on Symbionts Demands Synchronization.

Tsetse flies have socioeconomic significance as the obligate vector of multiple Trypanosoma parasites, the causative agents of Human and Animal African Trypanosomiases. Like many animals subsisting on a limited diet, microbial symbiosis is key to supplementing nutrient deficiencies necessary for metabolic, reproductive, and immune functions. Extensive studies on the microbiota in parallel to tsetse biology have unraveled the many dependencies partners have for one another. But far less is known mechanistically on how products are swapped between partners and how these metabolic exchanges are regulated, especially to address changing physiological needs. More specifically, how do metabolites contributed by one partner get to the right place at the right time and in the right amounts to the other partner? Epigenetics is the study of molecules and mechanisms that regulate the inheritance, gene activity and expression of traits that are not due to DNA sequence alone. The roles that epigenetics provide as a mechanistic link between host phenotype, metabolism and microbiota (both in composition and activity) is relatively unknown and represents a frontier of exploration. Here, we take a closer look at blood feeding insects with emphasis on the tsetse fly, to specifically propose roles for microRNAs (miRNA) and DNA methylation, in maintaining insect-microbiota functional homeostasis. We provide empirical details to addressing these hypotheses and advancing these studies. Deciphering how microbiota and host activity are harmonized may foster multiple applications toward manipulating host health, including identifying novel targets for innovative vector control strategies to counter insidious pests such as tsetse.

Immunofluorescent staining of cancer spheroids and fine-needle aspiration-derived organoids.

Our organoid generation technique has allowed for the development of downstream organoid applications. Here, we detail an accessible, straightforward protocol for immunofluorescent staining and imaging of thyroid cancer organoids, particularly those with tumor de-differentiation. Immunofluorescence is a powerful tool to help understand the localization of cell types within organoids and determine the interactions between those cells. As organoids have been shown to recapitulate patient tumor morphology, immunofluorescent staining and imaging of organoids allows for enhanced understanding of near in vivo structures. For complete details on the use and execution of this protocol, please refer to Lee et al. (2020) and Vilgelm et al. (2020).

Fine-Needle Aspiration-Based Patient-Derived Cancer Organoids.

Patient-derived cancer organoids hold great potential to accurately model and predict therapeutic responses. Efficient organoid isolation methods that minimize post-collection manipulation of tissues would improve adaptability, accuracy, and applicability to both experimental and real-time clinical settings. Here we present a simple and minimally invasive fine-needle aspiration (FNA)-based organoid culture technique using a variety of tumor types including gastrointestinal, thyroid, melanoma, and kidney. This method isolates organoids directly from patients at the bedside or from resected tissues, requiring minimal tissue processing while preserving the histologic growth patterns and infiltrating immune cells. Finally, we illustrate diverse downstream applications of this technique including in vitro high-throughput chemotherapeutic screens, in situ immune cell characterization, and in vivo patient-derived xenografts. Thus, routine clinical FNA-based collection techniques represent an unappreciated substantial source of material that can be exploited to generate tumor organoids from a variety of tumor types for both discovery and clinical applications.

Novel three-dimensional cultures provide insights into thyroid cancer behavior.

Thyroid cancer has the fastest growing incidence of any cancer in the United States, as measured by the number of new cases per year. Despite advances in tissue culture techniques, a robust model for thyroid cancer spheroid culture is yet to be developed. Using eight established thyroid cancer cell lines, we created an efficient and cost-effective 3D culture system that can enhance our understanding of in vivo treatment response. We found that all eight cell lines readily form spheroids in culture with unique morphology, size, and cytoskeletal organization. In addition, we developed a high-throughput workflow that allows for drug screening of spheroids. Using this approach, we found that spheroids from K1 and TPC1 cells demonstrate significant differences in their sensitivities to dabrafenib treatment that closely model expected patient drug response. In addition, K1 spheroids have increased sensitivity to dabrafenib when compared to monolayer K1 cultures. Utilizing traditional 2D cultures of these cell lines, we evaluated the mechanisms of this drug response, showing dramatic and acute changes in their actin cytoskeleton as well as inhibition of migratory behavior in response to dabrafenib treatment. Our study is the first to describe the development of a robust spheroid system from established cultured thyroid cancer cell lines and adaptation to a high-throughput format. We show that combining 3D culture with traditional 2D methods provides a complementary and powerful approach to uncover drug sensitivity and mechanisms of inhibition in thyroid cancer.

Size-mediated adaptive foraging: a host-selection strategy for insect parasitoids.

Foraging models are useful tools for generating predictions on predator-prey interactions, such as habitat or diet choice. However, the majority of studies attempting to explain adaptive behaviour using optimality criteria have assumed that there is no trait (e.g. size) variation among individual consumers or their prey. Hymenopteran parasitoids that attack the free-living stages of their host are an ideal system for studying the influence of body size on host selection because of the wide range of adult parasitoid sizes coupled with the defensive capabilities of their hosts. We report here our application of an experimentally parameterized host selection model to investigate the influence of parasitoid body size on the range of acceptable host instar classes. Using a demographic model, we compared the efficiency of parasitoids using an optimal host selection strategy against parasitoids using an indiscriminate host selection strategy over a range of different parasitoid body sizes. Net fitness accrual of parasitoids and the impact of host instar selection on aphid recruitment were assessed on different stage-structured aphid populations. Our results demonstrate that optimal host selection allows larger parasitoids to utilize a wider range of hosts. However, smaller parasitoids receive the greatest benefits from selecting hosts optimally by utilizing a restricted range of small, poorly defended hosts when they are abundant. We argue that the correlation between flexible host selection behaviour and adult body size may be a general phenomenon that applies to the majority of hymenopteran parasitoids that attack free-living, well-defended hosts. The potential of within-generation behavioural interactions to impact between-generation dynamics in host-parasitoid populations are discussed.

Sevoflurane general anesthesia: an alternative technique in the pediatric oral and maxillofacial surgery patient.

PURPOSE: The objective of this prospective descriptive study was to report our experience with an alternative general anesthetic technique, using sevoflurane for the pediatric patient, in an ambulatory setting. PATIENTS AND METHODS: Twenty consecutive pediatric patients participated over a 4-month period. A general anesthetic technique, using mask induction with sevoflurane and maintenance with a nasal trumpet as airway, was used throughout the surgical procedure. A standard anesthesia and recovery record was made for each patient; this included observations for untoward effects and complications. RESULTS: The average time for induction of anesthesia was 95 seconds, time from termination of sevoflurane to eye opening was 8 minutes, and duration of recovery was 30 minutes. Procedure time for each case did not exceed 10 minutes. Two patients had transient tachycardia. CONCLUSIONS: The results of this prospective descriptive study indicate that this technique is an effective and acceptable alternative to other modalities for the control of apprehension and fear in the pediatric patient in an ambulatory oral and maxillofacial facility.