Factors Influencing Opioid Receptor Signaling to Adenylyl Cyclase.

Opioids, such as morphine, signal through G protein-coupled receptors that are linked to adenylyl cyclase (AC)-inhibitory (Gi/o) heterotrimeric GTP-binding proteins. The transduction of signal from opioid receptor to G protein is dependent on the organization of proteins in the membrane and can be modulated by alterations in membrane lipids. This thesis provides evidence that mu-opioid receptors (MOR) and delta-opioid receptors (DOR) are differentially regulated by cholesterol. I have determined that cholesterol is required to stabilize MOR in a high-affinity conformation that couples to G proteins. Thus, cholesterol depletion attenuated the efficiency of MOR signaling to G proteins or AC. In contrast, DOR signaling was unaffected by removal of membrane cholesterol. Cholesterol depletion also attenuated the ability of MOR agonists to produce cellular adaptations leading to cAMP overshoot. This was true of heterologously expressed MOR in HEK293 cells, and endogenously expressed MOR in human neuroblastoma SH-SY5Y cells. Cholesterol may modulate MOR signaling by a direct interaction with the receptor or by formation of cholesterol-enriched membrane microdomains. Indeed, a portion of cell surface MOR was associated with markers of cholesterol-enriched membrane microdomains; however, these studies do not confirm that this association is responsible for the effect of cholesterol depletion on MOR signaling. In contrast, DOR was not associated with membrane microdomain markers, even after agonist treatment. MOR and DOR are thus likely not compartmentalized together within cholesterol-enriched membrane microdomains. Regardless, MOR and DOR in SH-SY5Y cells were observed to share a common pool of G proteins and AC, which resulted in occlusion of DOR agonist responses in the presence of maximally effective concentrations of MOR agonist. Moreover, this was not limited to MOR and DOR, as all Gi/o-coupled receptors endogenously expressed in SH-SY5Y cells were able to access a shared pool of AC. After chronic administration of MOR agonist, the addition of agonists to DOR, alpha2-adrenergic receptors (alpha2AR) and nociceptin/OFQ peptide receptors (NOPr) reduced the expression of MOR-mediated cAMP overshoot. Given the co-expression of alpha2AR and NOPr on MOR-containing neurons, the ability of agonists to these receptors to reduce MOR-mediated cAMP overshoot has implications in the treatment of opioid withdrawal symptoms.Ph.D.PharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/77937/1/elsawyer_1.pd

Parental Expressed Emotion During Two Forms of Family-Based Treatment for Adolescent Anorexia Nervosa.

High parental expressed emotion (EE), reflected by criticism or emotional over-involvement, has been related to poorer outcome in family-based treatment (FBT) for adolescent anorexia nervosa. This study assessed EE in 89 mothers and 64 fathers at baseline and end of treatment in a randomised trial comparing conjoint FBT to parent-focused FBT (PFT). Compared with conjoint FBT, PFT was associated with a decrease in maternal criticism, regardless of adolescent remission. Furthermore, an increase in maternal criticism was more likely to be observed in conjoint FBT (80%) than PFT (20%, p = 0.001). Adolescents of mothers who demonstrated an increase in EE, or remained high in EE, were less likely to remit compared with adolescents for whom EE decreased or remained low (33% and 0% vs. 43% and 50%, p = 0.03). There were no significant effects for paternal EE. The results highlight the importance of considering EE when implementing FBT for adolescents with anorexia nervosa. Copyright © 2017 John Wiley & Sons, Ltd and Eating Disorders Association

Parental Expressed Emotion During Two Forms of Family-Based Treatment for Adolescent Anorexia Nervosa.

High parental expressed emotion (EE), reflected by criticism or emotional over-involvement, has been related to poorer outcome in family-based treatment (FBT) for adolescent anorexia nervosa. This study assessed EE in 89 mothers and 64 fathers at baseline and end of treatment in a randomised trial comparing conjoint FBT to parent-focused FBT (PFT). Compared with conjoint FBT, PFT was associated with a decrease in maternal criticism, regardless of adolescent remission. Furthermore, an increase in maternal criticism was more likely to be observed in conjoint FBT (80%) than PFT (20%, p = 0.001). Adolescents of mothers who demonstrated an increase in EE, or remained high in EE, were less likely to remit compared with adolescents for whom EE decreased or remained low (33% and 0% vs. 43% and 50%, p = 0.03). There were no significant effects for paternal EE. The results highlight the importance of considering EE when implementing FBT for adolescents with anorexia nervosa. Copyright © 2017 John Wiley & Sons, Ltd and Eating Disorders Association

Phenotyping grapevine red blotch virus and grapevine leafroll-associated viruses before and after symptom expression through machine-learning analysis of hyperspectral images.

INTRODUCTION: Grapevine leafroll-associated viruses (GLRaVs) and grapevine red blotch virus (GRBV) cause substantial economic losses and concern to North Americas grape and wine industries. Fast and accurate identification of these two groups of viruses is key to informing disease management strategies and limiting their spread by insect vectors in the vineyard. Hyperspectral imaging offers new opportunities for virus disease scouting. METHODS: Here we used two machine learning methods, i.e., Random Forest (RF) and 3D-Convolutional Neural Network (CNN), to identify and distinguish leaves from red blotch-infected vines, leafroll-infected vines, and vines co-infected with both viruses using spatiospectral information in the visible domain (510-710nm). We captured hyperspectral images of about 500 leaves from 250 vines at two sampling times during the growing season (a pre-symptomatic stage at veraison and a symptomatic stage at mid-ripening). Concurrently, viral infections were determined in leaf petioles by polymerase chain reaction (PCR) based assays using virus-specific primers and by visual assessment of disease symptoms. RESULTS: When binarily classifying infected vs. non-infected leaves, the CNN model reaches an overall maximum accuracy of 87% versus 82.8% for the RF model. Using the symptomatic dataset lowers the rate of false negatives. Based on a multiclass categorization of leaves, the CNN and RF models had a maximum accuracy of 77.7% and 76.9% (averaged across both healthy and infected leaf categories). Both CNN and RF outperformed visual assessment of symptoms by experts when using RGB segmented images. Interpretation of the RF data showed that the most important wavelengths were in the green, orange, and red subregions. DISCUSSION: While differentiation between plants co-infected with GLRaVs and GRBV proved to be relatively challenging, both models showed promising accuracies across infection categories

Opioid Management in the Dying Child With Addiction

The dramatic increases of opioid use and misuse in the past 15 years have resulted in a focus on the responsible and judicious use of opioids. In this Ethics Rounds, the commentators analyze the case of a 16-year-old girl with lymphoma and opioid misuse whose caregiver may have diverted her opioids. She is now at the end of life and prefers to die at home. The commentators, oncologists, palliative care providers, ethicists, and a medical student agree that supporting the patient\u27s goals and practicing good opioid stewardship are not incompatible. They identify additional information that would be required to analyze the case more fully such as the nature of the evidence for misuse and diversion and whether bias inadvertently contributed to these concerns. They agree that multimodal analgesia, including but not limited to opioids, is important. Safeguards could include a contract, directly observed therapy, and/or urine drug screens. Supervision or removal of a caregiver diverting medication or admission of the patient misusing medications would be alternatives if the initial plan was unsuccessful. Such patient-centered care requires well-developed substance misuse treatment, pain management, and home hospice that are adequately reimbursed

Active DNA unwinding and transport by a membrane-adapted helicase nanopore.

Nanoscale transport through nanopores and live-cell membranes plays a vital role in both key biological processes as well as biosensing and DNA sequencing. Active translocation of DNA through these nanopores usually needs enzyme assistance. Here we present a nanopore derived from truncated helicase E1 of bovine papillomavirus (BPV) with a lumen diameter of c.a. 1.3 nm. Cryogenic electron microscopy (cryo-EM) imaging and single channel recording confirm its insertion into planar lipid bilayer (BLM). The helicase nanopore in BLM allows the passive single-stranded DNA (ssDNA) transport and retains the helicase activity in vitro. Furthermore, we incorporate this helicase nanopore into the live cell membrane of HEK293T cells, and monitor the ssDNA delivery into the cell real-time at single molecule level. This type of nanopore is expected to provide an interesting tool to study the biophysics of biomotors in vitro, with potential applications in biosensing, drug delivery and real-time single cell analysis