Identifying and training deep learning neural networks on biomedical-related datasets.

This manuscript describes the development of a resources module that is part of a learning platform named 'NIGMS Sandbox for Cloud-based Learning' https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox at the beginning of this Supplement. This module delivers learning materials on implementing deep learning algorithms for biomedical image data in an interactive format that uses appropriate cloud resources for data access and analyses. Biomedical-related datasets are widely used in both research and clinical settings, but the ability for professionally trained clinicians and researchers to interpret datasets becomes difficult as the size and breadth of these datasets increases. Artificial intelligence, and specifically deep learning neural networks, have recently become an important tool in novel biomedical research. However, use is limited due to their computational requirements and confusion regarding different neural network architectures. The goal of this learning module is to introduce types of deep learning neural networks and cover practices that are commonly used in biomedical research. This module is subdivided into four submodules that cover classification, augmentation, segmentation and regression. Each complementary submodule was written on the Google Cloud Platform and contains detailed code and explanations, as well as quizzes and challenges to facilitate user training. Overall, the goal of this learning module is to enable users to identify and integrate the correct type of neural network with their data while highlighting the ease-of-use of cloud computing for implementing neural networks. This manuscript describes the development of a resource module that is part of a learning platform named ``NIGMS Sandbox for Cloud-based Learning'' https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox [1] at the beginning of this Supplement. This module delivers learning materials on the analysis of bulk and single-cell ATAC-seq data in an interactive format that uses appropriate cloud resources for data access and analyses.

Using focused ethnography to inform biomedical research infrastructure enhancement at primarily undergraduate institutions.

The National Institutes of Health (NIH) supports 24 IDeA Networks of Biomedical Research Excellence (INBRE) Programs that help develop university-based biomedical research capacity in states that historically receive low levels of extramural grant support. To assess the effectiveness of the Arkansas INBRE in meeting its biomedical research capacity-building goals, we evaluated how the context (i.e., local and institutional settings) at two undergraduate institutions impacted variability in science faculty use of program resources. Data were collected by in-depth interviews with faculty and administrators (N = 9), focused observations, a review of Arkansas INBRE databases, and internet searches. Content analysis was used to code interview transcripts and field notes, and then qualitative data were integrated with data from databases and internet searches to construct two institutional case summaries. Constant comparison was used to identify similarities and differences between the institutions that helped to explain variability in how frequently faculty used Arkansas INBRE resources, including an enrollment crisis at undergraduate institutions in the United States and the presence or absence of a robust research culture at each institution. These findings were used to suggest program improvements (e.g., classroom-based research) that could further strengthen biomedical research capacity in Arkansas. As some barriers to program effectiveness are likely found in other IDeA-eligible states, improvements suggested for the Arkansas INBRE could apply to INBRE programs elsewhere.NEW & NOTEWORTHY This article describes results from an approach to program evaluation (i.e., focused ethnography) that has not been previously used to evaluate grant mechanisms. This "experience near" approach, which involved qualitative interviews and firsthand observations, lent valuable insights into how broader and institutional contexts at two primarily undergraduate institutions hindered or facilitated use of Arkansas INBRE resources. The insights gained can be used to enhance the Arkansas INBRE, which aims to strengthen the statewide biomedical infrastructure.

COVID-19 Booster Uptake: Are Hesitant Adopters Less Likely to Get a Booster Shot Than Nonhesitant Adopters?

The main objective of this study was to assess whether hesitancy toward receiving the initial COVID-19 vaccine was associated with uptake of the COVID-19 booster several months after it became available to all US adults. We ask whether hesitancy toward the initial COVID-19 vaccine was significantly associated with lower odds of COVID-19 booster uptake among adults. We test this association within the context of the highly rural state of Arkansas. By January 2022, the US had set a global record of nearly 1 million daily cases. The purpose of this study was to advance our understanding of vaccine hesitancy among those who have already received a dose of the COVID-19 vaccine and how that hesitancy may shape COVID-19 booster uptake. We analyzed data from a random sample survey of Arkansan adults (N = 2,201) between March 1 and March 28, 2022 and constrained our analytical sample to those who had received a vaccine (N = 1,649). Nearly two-thirds of vaccinated Arkansas residents had received a COVID-19 booster. Hesitancy was common even among vaccinated individuals and was significantly associated with reduced odds of COVID-19 booster uptake, even after controlling for other factors. Findings provide further support for conceptualizing vaccine hesitancy as an attitude related to-but separate from-the behavior of vaccination, as opposed to conflating vaccination with being nonhesitant. Public health interventions aimed at increasing COVID-19 booster uptake should pay attention to vaccine hesitancy indicated at the initiation of the series and should not ignore the vaccinated as an important population to target for intervention.

Impact of the INBRE summer student mentored research program on undergraduate students in Arkansas.

The Institutional Development Award (IDeA) program, housed within the National Institute for General Medical Sciences, administers the Networks of Biomedical Research Excellence (INBRE) as a strategic mission to broaden the geographic distribution of National Institutes of Health (NIH) funding within the United States. Undergraduate summer student mentored research programs (SSMRP) are a common feature of INBRE programs and are designed to increase undergraduate student interest in research careers in the biomedical sciences. Little information is available about student perspectives on how these programs impact their choices relative to education and careers. Therefore, we conducted qualitative interviews with 20 participants from the Arkansas INBRE SSMRP in the years 2002-2012. Each telephone interview lasted 30-45 min. An interview guide with a broad "grand tour" question was used to elicit student perspectives on SSMRP participation. Interviews were digitally recorded, then transcribed verbatim, and the transcript checked for accuracy. Content analysis and constant comparison were used to identify nine themes that were grouped into three temporal categories: before, during, and after the SSMRP experience. Students viewed the experience as positive and felt it impacted their career choices. They emphasized the value of mentoring in the program, and some reported maintaining a relationship with the mentor after the summer experience ended. Students also valued learning new laboratory and presentation skills and felt their research experience was enhanced by meeting students and scientists with a wide range of career interests. These data suggest that the Arkansas INBRE and the NIH IDeA program are successfully meeting the goal of increasing interest in research among undergraduates.

A possible mechanism contributing to the synergistic action of vasotocin (VT) and corticotropin-releasing hormone (CRH) receptors on corticosterone release in birds.

Arginine vasotocin (AVT) and corticotropin-releasing hormone (CRH) are two neuronal regulators in the hypothalamic-pituitary-adrenal (HPA) axis that modulate biological responses to stress in avian species. When AVT and CRH are administered together in vitro or in vivo, levels of adrenocorticotropic hormone (ACTH) or plasma corticosterone (CORT) are released, respectively, in a synergistic manner. The underlying mechanism of this greater than additive stress response was investigated by expressing the vasotocin receptor type 2 (VT2R) and CRH receptor type 1 (CRH-R1), both G-protein coupled receptors, in HeLa cells. Fluorescence resonance energy transfer (FRET) analysis provided the evidence for heterodimerization of the VT2R/CRH-R1 in the presence of their respective ligands, AVT and CRH. The VT2R and CRH-R1 were tagged at the C-terminal ends with either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), and a VT2R chimera was constructed by replacing the fourth transmembrane region (TM4) of the VT2R with TM-IV of the ß2-adrenergic receptor (ß2AR). When VT2R/ß2AR chimera and CRH-R1 were expressed in HeLa cells, heterodimerization was partly disrupted. Taken together, these data indicate that TM-IV of the VT2R may provide an important interface for effective receptor dimerization, suggesting that direct molecular interaction between VT2R and CRH-R1 receptors plays a role in mediating an enhanced interaction between these two receptors. Their interaction at the anterior pituitary level may potentiate the endocrine output of the avian HPA system.

Social processes, practical issues, and COVID-19 vaccination among hesitant adults.

INTRODUCTION: The purpose of this study is to examine relationships between COVID-19 vaccination, social processes, and the practical issues of healthcare coverage and workplace requirements. We examine these relationships among individuals who expressed some degree of hesitancy towards receiving the vaccine. Assessing relationships between COVID-19 vaccination, social processes, and practical issues among vaccine-hesitant individuals has implications for public health policy and intervention. METHODS: We analyzed weighted data from a random sample phone survey of Arkansas adults (N = 2,201) between March 1st and March 28th, 2022 and constrained our analytical sample to those who had reported some degree of vaccine hesitancy (N = 1,251). Statistical analyses included weighted and unweighted descriptive statistics, weighted bivariate logistic regressions, and a weighted multivariate logistic regression to obtain adjusted odds ratios for COVID-19 vaccination. RESULTS: More than two-thirds (62.5 %) of respondents were vaccinated, despite their hesitancy. Adjusted odds of COVID-19 vaccination were greater among Black (OR = 2.55; 95 % CI[1.63, 3.97]) and Hispanic respondents (OR = 2.46; 95 % CI[1.53, 3.95]), respondents whose healthcare provider recommended vaccination (OR = 2.50; 95 % CI[1.66, 3.77]), and as perceptions of vaccination coverage (OR = 2.04; 95 % CI[1.71, 2.43]) and subjective social status increased (OR = 1.10; 95 % CI[1.01, 1.19]). Adjusted odds of COVID-19 vaccination were greater among respondents with a workplace that recommended (OR = 1.96; 95 % CI[1.03, 3.72]) or required vaccination (OR = 12.62; 95 % CI[4.76, 33.45]) and among respondents who were not employed (OR = 1.82; 95 % CI[1.10, 3.01]) compared to those whose workplace did not recommend or require COVID-19 vaccination. CONCLUSIONS: Some hesitant individuals become vaccinated despite their hesitancy-a group we refer to as "hesitant adopters." Social processes and practical issues are important correlates of vaccination among those who are hesitant. Workplace requirements appear to be of particular importance for vaccination among hesitant individuals. Provider recommendations, norms, social status, and workplace policies may be effective points of intervention among those who express vaccine hesitancy.

Development of a clinical and translational research curriculum for undergraduate students.

Introduction: Research participation during undergraduate years has a powerful influence on career selection and attitudes toward scientific research. Most undergraduate research programs in academic health centers are oriented toward basic research or address a particular disease focus or research discipline. Undergraduate research programs that expose students to clinical and translational research may alter student perceptions about research and influence career selection. Methods: We developed an undergraduate summer research curriculum, anchored upon a clinical and translational research study developed to address a common unmet needs in neonatal nurseries (e.g., assessment of neonatal opioid withdrawal syndrome). Program topics reflected the cross-disciplinary expertise that contributed to the development of this "bedside to bench" study, including opioid addiction, vulnerable populations, research ethics, statistics, data collection and management, assay development, analytical laboratory analysis, and pharmacokinetics. The curriculum was delivered through three offerings over 12 months, using Zoom video-conferencing due to restrictions imposed by the COVID-19 pandemic. Results: Nine students participated in the program. Two-thirds reported the course enhanced their understanding of clinical and translational research. Over three-quarters reported the curriculum topics were very good or excellent. In open-ended questions, students reported that the cross-disciplinary nature of the curriculum was the strongest aspect of the program. Conclusion: The curriculum could be readily adapted by other Clinical and Translational Science Award programs seeking to provide clinical and translational research-oriented programs to undergraduate students. Application of cross-disciplinary research approaches to a specific clinical and translational research question provides students with relevant examples of translational research and translational science.

Effects of the ß-agonist, isoprenaline, on the down-regulation, functional responsiveness and trafficking of ß2-adrenergic receptors with N-terminal polymorphisms.

The ß2-AR (ß2-adrenergic receptor) is an important target for respiratory and CVD (cardiovascular disease) medications. Clinical studies suggest that N-terminal polymorphisms of ß2-AR may act as disease modifiers. We hypothesized that polymorphisms at amino acids 16 and 27 result in differential trafficking and down-regulation of ß2-AR variants following ß-agonist exposure. The functional consequences of the four possible combinations of these polymorphisms in the human ß2-AR (designated ß2-AR-RE, ß2-AR-GE, ß2-AR-RQ and ß2-AR-GQ) were studied using site-directed mutagenesis and recombinant expression in HEK-293 cells (human embryonic kidney cells). Ligand-binding assays demonstrated that after 24 h exposure to 1 µM isoprenaline, isoforms with Arg16 (ß2-AR-RE and ß2-AR-RQ) underwent increased down-regulation compared with isoforms with Gly16 (ß2-AR-GE and ß2-AR-GQ). Consistent with these differences in down-regulation between isoforms, prolonged isoprenaline treatment resulted in diminished cAMP response to subsequent isoprenaline challenge in ß2-AR-RE relative to ß2-AR-GE. Confocal microscopy revealed that the receptor isoforms had similar co-localization with the early endosomal marker EEA1 following isoprenaline treatment, suggesting that they had similar patterns of internalization. None of the isoforms exhibited significant co-localization with the recycling endosome marker Rab11 in response to isoprenaline treatment. Furthermore, we found that prolonged isoprenaline treatment led to a higher degree of co-localization of ß2-AR-RE with the lysosomal marker LAMP1 (lysosome-associated membrane protein 1) compared with that of ß2-AR-GE. Taken together, these results indicate that a mechanism responsible for differential responses of these receptor isoforms to the ß-agonist involves differences in the efficiency with which agonist-activated receptors are trafficked to the lysosomes for degradation, or differences in degradation in the lysosomes.

Age-dependent expression of AVT and its oxytocic-like receptor VT3 in the shell gland of Japanese quail, Coturnix coturnix japonica.

The avian neurohypophyseal hormone AVT is an important regulatory hormone involved in many physiological processes including oviposition; an age-related phenomenon, through its action on the shell gland. In this study, immunohistochemistry and in situ hybridization was performed to study the expression of immunoreactive (ir) AVT and its oxytocic-like receptor VT3 transcript in the ovary/shell gland of Japanese quail representing sexually immature, mature and old condition. Our results indicate that ir-AVT is present in the ovary of sexually active adult only, but in the shell gland it is observed in both sexually active adult and sexually quiescent old quail. Further, VT3 gene transcript although not detected in the shell gland of sexually immature birds, has been found abundantly in the myometrium of shell gland of sexually active adult quail with a slight decrease in old birds. It is concluded that in addition to the ovarian function and shell gland activity, the expression of AVT and VT3 receptor in the shell gland also varies with the age dependent reproductive/egg laying performance of the Japanese quail. Our findings also suggest that (i) local synthesis of AVT and the expression of its oxytocic-like VT3 receptors is estrogen dependent and (ii) shell gland AVT upregulates its VT3 receptor (paracrine role) in advance to facilitate the role of neurohypophyseal AVT during oviposition.

Osmotic stress induced alteration in the expression of arginine vasotocin receptor VT2 in the pituitary gland and adrenal function of domestic fowl.

The role of arginine vasotocin in the regulation of the pituitary-adrenal axis of domestic fowl was analyzed by studying the expression of its recently cloned pituitary receptor VT2 and adrenal activity following osmotic stress. Four days of water deprivation induced an increase in plasma osmolality-a known stimulator of AVT synthesis and release from hypothalamic magnocellular neurons. Water deprivation also decreased pituitary mRNA levels for both the VT2 receptor and for pro-opiomelanocortin (POMC). Despite a decrease in the expression of VT2 mRNA, immunoreactive-VT2 receptor levels in the pituitary increased, suggesting a possible role for post-transcriptional mechanisms in the regulation of this receptor. Further, adrenal activity (as judged by adrenal weight, cholesterol content, 3beta hydroxysteroid dehydrogenase, cortical cord width and cortico-medullary ratio) showed stimulation in water-deprived chicken as compared to control. On the basis of present findings, it is concluded that water deprivation down regulates the mRNA expression of AVT receptor VT2 as well as POMC but stimulates adrenal function. It is also suggested that in addition to the release of magnocellular AVT into the neurohypophysis to act as antidiuretic hormone following water deprivation, AVT may also modulate HPA axis to cope with the osmotic stress.

Corticosterone- or metapyrone-induced alterations in adrenal function and expression of the arginine vasotocin receptor VT2 in the pituitary gland of domestic fowl, Gallus gallus.

The avian neurohypophyseal hormone arginine vasotocin (AVT) is known to be involved in the regulation of adrenocorticotropin (ACTH) release by interacting with the VT2 receptor (VT2R), which is homologous to the mammalian vasopressin V1b receptor (V1bR). To study the role of glucocorticoid in the expression and regulation of the VT2R, corticosterone (1 or 5mg/bird/day) or metapyrone (10 or 50mg/kg body weight/day) were administered daily for 8 days to white leghorn chickens. While low doses were ineffective, a high dose of corticosterone upregulated, while metapyrone downregulated, pituitary VT2R mRNA expression and ir-VT2 in the cephalic lobe of the anterior pituitary. Further, although no change was observed in the expression of POMC mRNA, adrenal activity (as judged by the changes in total cholesterol, 3beta HSD, cortical cord width and cortico-medullary ratio) exhibited suppression or stimulation following treatment with corticosterone or metapyrone, respectively. In view of the classical negative feedback effect of glucocorticoids at the level of hypothalamic CRH neurons and pituitary corticotrophs, high corticosterone level-induced suppression of the CRH-ACTH axis may have been masked by VT2R-mediated stimulation of corticotrophs, and hence the POMC mRNA level did not change. The same argument could be used for metapyrone. It is concluded that expression of the VT2 receptor is regulated by glucocorticoids in chicken. These findings confirm a role for AVT, mediated by the VT2 receptor, in regulating ACTH secretion during stress and suggest that corticotroph VT2 receptor levels may be dynamically regulated depending on the HPA axis activity.

Characterization of a panel of six beta2-adrenergic receptor antibodies by indirect immunofluorescence microscopy.

BACKGROUND: The beta2-adrenergic receptor (beta2AR) is a primary target for medications used to treat asthma. Due to the low abundance of beta2AR, very few studies have reported its localization in tissues. However, the intracellular location of beta2AR in lung tissue, especially in airway smooth muscle cells, is very likely to have a significant impact on how the airways respond to beta-agonist medications. Thus, a method for visualizing beta2AR in tissues would be of utility. The purpose of this study was to develop an immunofluorescent labeling technique for localizing native and recombinant beta2AR in primary cell cultures. METHODS: A panel of six different antibodies were evaluated in indirect immunofluorescence assays for their ability to recognize human and rat beta2AR expressed in HEK 293 cells. Antibodies capable of recognizing rat beta2AR were identified and used to localize native beta2AR in primary cultures of rat airway smooth muscle and epithelial cells. beta2AR expression was confirmed by performing ligand binding assays using the beta-adrenergic antagonist [3H] dihydroalprenolol ([3H]DHA). RESULTS: Among the six antibodies tested, we identified three of interest. An antibody developed against the C-terminal 15 amino acids of the human beta2AR (Ab-Bethyl) specifically recognized human but not rat beta2AR. An antibody developed against the C-terminal domain of the mouse beta2AR (Ab-sc570) specifically recognized rat but not human beta2AR. An antibody developed against 78 amino acids of the C-terminus of the human beta2AR (Ab-13989) was capable of recognizing both rat and human beta2ARs. In HEK 293 cells, the receptors were predominantly localized to the cell surface. By contrast, about half of the native rat beta2AR that we visualized in primary cultures of rat airway epithelial and smooth muscle cells using Ab-sc570 and Ab-13989 was found inside cells rather than on their surface. CONCLUSION: Antibodies have been identified that recognize human beta2AR, rat beta2AR or both rat and human beta2AR. Interestingly, the pattern of expression in transfected cells expressing millions of receptors was dramatically different from that in primary cell cultures expressing only a few thousand native receptors. We anticipate that these antibodies will provide a valuable tool for evaluating the expression and trafficking of beta2AR in tissues.

Transmembrane domain IV of the Gallus gallus VT2 vasotocin receptor is essential for forming a heterodimer with the corticotrophin releasing hormone receptor.

Corticotropin releasing hormone receptor (CRHR) and the VT2 arginine vasotocin receptor (VT2R) are vital links in the hypothalamic-pituitary-adrenal axis that enable a biological response to stressful stimuli in avian species. CRHR and VT2R are both G-protein coupled receptors (GPCRs), and have been shown by us to form a heterodimer via fluorescent resonance energy transfer (FRET) analysis in the presence of their respective ligands, corticotrophin releasing hormone (CRH) and arginine vasotocin (AVT). The dimerization interface of the heterodimer is unknown, but computational analyses predict transmembrane domains (TMs) as likely sites of the interaction. We constructed chimerical VT2Rs, tagged at the C-terminal ends with either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), by replacing the fourth transmembrane region (TM4) of VT2R with TM4 of the beta2-adrenergic receptor (beta2AR). The VT2R/beta2AR chimeras were expressed in HeLa cells and proper trafficking is confirmed by observing cell membrane localization using confocal microscopy. VT2R/beta2AR-YFP chimera functionality was confirmed with a Fura-2 acetoxymethyl ester (Fura-2AM) assay. FRET analysis was then performed on VT2/beta2AR-chimera/CRHR pairs, and the calculated distance was observed to be >10 nm apart, indicating that heterodimerization was partly disrupted by mutating TM4 of the VT2R. Therefore, TM4 may form one region of the possible dimerization interfaces between the VT2R and CRHR.

Heterooligomerization between vasotocin and corticotropin-releasing hormone (CRH) receptors augments CRH-stimulated 3',5'-cyclic adenosine monophosphate production.

In birds, ACTH release from the anterior pituitary gland during stress is controlled by CRH and arginine vasotocin (AVT). Using 5-wk-old male chicks, simultaneous iv injections of CRH and AVT were found to result in a greater than additive increase in plasma corticosterone levels compared with that obtained with individual administration of either peptide hormone. In order to investigate molecular mechanisms underlying this observation, the chicken CRH receptor (CRHR) and vasotocin VT2 receptor (VT2R) were fused to cyan and yellow fluorescent proteins and expressed in HeLa cells. The resulting CRHR and VT2R fusion proteins were expressed appropriately in the plasma membrane and were found to couple to downstream signal transduction pathways. Quantitative fluorescence resonance energy transfer (FRET) analysis was used to determine whether the CRHR and VT2R formed heterodimers. In the absence of CRH and AVT, the FRET efficiency was 15-18%, and the distance between receptors was 5-6 nm. Treatment of the cells that expressed both cyan fluorescent protein-CRHR and yellow fluorescent protein-VT2R with CRH or AVT alone did not lead to a significant change in the FRET efficiency. However, simultaneous addition of these hormones increased the efficiency of the FRET signal and decreased the distance between the two receptors. In HeLa cells expressing both CRHR and VT2R, treatment with CRH and AVT resulted in a significant increase in cAMP production over that with CRH alone, indicating that heterodimer formation may enhance the ability of the CRHR to activate downstream signal transduction.

Effect of estrogen and its antagonist on the expression of arginine vasotocin (AVT) and its oxytocic-like receptor VT3 in the shell gland of Japanese quail, Coturnix coturnix japonica.

Avian neurohypophysial hormone arginine vasotocin (AVT) is known to regulate shell gland contractility during oviposition. While studying the role of estrogen in the expression and regulation of AVT and its oxytocic-like receptor VT3, using in situ hybridization and immunohistochemistry, it was observed that the expression of AVT and its receptor was not detected in the shell gland of sexually immature Japanese quail. However, administration of estrogen to these birds not only stimulates the growth and activity (as assessed by increased mucosal fold length, total protein content and alkaline phosphatase level) of the shell gland but also upregulates the expression of AVT and VT3. Further, administration of estrogen antagonist tamoxifen to sexually mature bird shows opposite results. On the other hand, localization of ir-AVT, observed in the ovary of sexually mature bird, was not detected in the estrogen treated sexually immature quail. It is concluded that estrogen not only affects the growth and differentiation of avian oviduct, but also regulates the expression of shell gland AVT and its receptor VT3. Present findings suggest that the locally synthesized AVT acts in a paracrine way to upregulate VT3 receptor and thus facilitates the endocrine function of neurohypophysial AVT during oviposition.

Impact of estrogen and photoperiod on arginine vasotocin and VT3 receptor expression in the shell gland of quail.

Role of estrogen and photoperiod is well-established in avian reproduction. In addition, the distribution and the expression of arginine vasotocin (AVT) and its receptor VT3 to ensure reproductive/breeding conditions in Japanese quail influenced by them has been the main focus of this review. To consider this aspect the mRNA expression of VT3 receptor and its ligand AVT in the shell gland has been emphasized. In birds, AVT performs a dual role as an anti-diuretic hormone and the functions accomplished by oxytocin in mammals. The physiological actions of AVT in birds are mediated through its diverse receptor subtypes VT1, VT2, VT3 and VT4.  Dynamic alteration of VT3 expression during different reproductive and photo-sexual conditions of quail can be modulated by estrogen. In addition to the endocrine effect of AVT, the shell gland is complemented by its paracrine action via its receptors. Evidences indicate that the expression of shell gland AVT modulated by estrogen appears to play a priming role by modulating the availability of VT3 receptor for the required action of neurohypophysial AVT during oviposition.

The cloned avian neurohypophysial hormone receptors.

Arginine vasotocin (AVT), a neurohypophysial hormone, has many essential functions in birds including the regulation of salt and fluid balance, blood pressure, the stress response and a variety of behaviors. In addition, AVT controls reproductive functions in birds that are served by oxytocin in mammals. In the following review, we examine the functions of AVT in birds with an emphasis on the present state of knowledge concerning the cloned receptors for this important hormone. Receptor and gene structure, signal transduction mechanisms and expression pattern are all discussed. Finally, we explore the phylogenetic relationships between the cloned avian receptors and other vertebrate and invertebrate neurohypophysial hormone receptors.

Molecular cloning of an oxytocin-like receptor expressed in the chicken shell gland.

The avian homologs of arginine vasopressin (AVP) and oxytocin (OT) are arginine vasotocin (AVT) and mesotocin (MT), respectively. In birds, AVT shares many of the functions of AVP including regulation of fluid balance, blood pressure regulation and the stress response. AVT also plays an oxytocin-like reproductive role in birds by stimulating uterine (shell gland) contraction during oviposition. The role of MT in avian reproduction is not clear. Here, we report the cloning of a third neuropeptide receptor in the chicken (Gallus gallus). Parsimony analysis reveals that the new receptor has highest homology to mammalian OT receptors and the MT receptors of non-mammalian vertebrates. Moreover, the receptor bears far less homology to the two avian VT receptors that have been cloned. Reverse transcription-polymerase chain reaction and in in situ hybridization analyses reveal the receptor is expressed in both the endometrium and myometrium of the shell gland. The expression pattern and high homology to OT receptors suggest that the receptor may stimulate myometrial contraction and therefore play a critical role in oviposition.

Molecular cloning and functional characterization of a vasotocin receptor subtype expressed in the pituitary gland of the domestic chicken (Gallus domesticus): avian homolog of the mammalian V1b-vasopressin receptor.

The neurohypophysial hormone arginine vasotocin (AVT) stimulates adrenocorticotropin hormone (ACTH) secretion from the avian anterior pituitary gland resulting in increased adrenal secretion of corticosterone in response to stress. Here, we report molecular cloning and functional characterization of a gene encoding an AVT receptor subtype, designated the VT2 receptor, that may mediate the stimulatory effect of AVT on ACTH secretion in birds. The open reading frame predicts a 425 amino acid polypeptide that includes seven segments of 19 to 24 hydrophobic amino acids, typical of guanine nucleotide-protein coupled receptors. Phylogenetic analysis revealed that the VT2 receptor shares highest identity with the mammalian V1b-vasopressin receptor subtype. Expressed VT2 receptors in COS7 cells mediate AVT-induced phosphatidylinositol turnover and Ca(2+) mobilization. In the domestic chicken, expression of VT2 receptor gene transcripts is limited to the pituitary gland. Based on similarities in sequence, site of expression and coupled signal transduction pathways, we conclude that the VT2 receptor is the avian homolog of the mammalian V1b-vasopressin receptor, and therefore may play an important role in the avian stress response.

Enhanced beta2-adrenergic receptor (beta2AR) signaling by adeno-associated viral (AAV)-mediated gene transfer.

BACKGROUND: Beta2-adrenergic receptors (beta2AR) play important regulatory roles in a variety of cells and organ systems and are important therapeutic targets in the treatment of airway and cardiovascular disease. Prolonged use of beta-agonists results in tolerance secondary to receptor down-regulation resulting in reduced therapeutic efficiency. The purpose of this work is to evaluate the signaling capabilities of the beta2AR expressed by a recombinant adeno-associated viral (AAV) vector that also included an enhanced green fluorescent protein (EGFP) gene (AAV-beta2AR/EGFP). RESULTS: By epifluorescence microscopy, approximately 40% of infected HEK 293 cells demonstrated EGFP expression. beta2AR density measured with [3H]dihydroalprenolol ([3H]DHA) increased either 13- or 77-fold in infected cells compared to mock infected controls depending on the culture conditions used. The [3H]DHA binding was to a single receptor population with a dissociation constant of 0.42 nM, as would be expected for wild-type beta2AR. Agonist competition assays with [3H]DHA showed the following rank order of potency: isoproterenol>epinephrine> norepinephrine, consistent with beta2AR interaction. Isoproterenol-stimulated cyclic AMP levels were 5-fold higher in infected cells compared to controls (314 +/- 43 vs. 63.4 +/- 9.6 nmol/dish; n = 3). Receptor trafficking demonstrated surface expression of beta2AR with vehicle treatment and internalization following isoproterenol treatment. CONCLUSIONS: We conclude that HEK 293 cells infected with AAV-beta2AR/EGFP effectively express beta2AR and that increased expression of these receptors results in enhanced beta2AR signaling. This method of gene transfer may provide an important means to enhance function in in vivo systems.