Inhibiting miR-618 Promotes Keratinocytes Proliferation and Migration to Enhance Wound Healing in Mice.

The delay in wound healing caused by chronic wounds or pathological scars is a pressing issue in clinical practice, imposing significant economic and psychological burdens on patients. In particular, with the aging of the population and the increasing incidence of diseases such as diabetes, impaired wound healing is one of the growing health problems. MicroRNA (miRNA) plays a crucial role in wound healing and regulates various biological processes. Our results show that miR-618 was significantly upregulated during the inflammatory phase of wound healing.Subsequently, miR-618 promotes the secretion of pro-inflammatory cytokines and regulates the proliferation and migration of keratinocytes. Mechanistically, miR-618 binds to the target gene-Atp11b and inhibits the PI3K-Akt signaling pathway, inhibiting the epithelial-mesenchymal transition (EMT) of keratinocytes. In addition, the PI3K-Akt signaling pathway induces the enrichment of nuclear miR-618, and miR-618 binds to the promoter of Lin7a to regulate gene transcription. Intradermal injection of miR-618 antagomir around full-thickness wounds in peridermal mice effectively accelerates wound closure compared to control. In conclusion, miR-618 antagomir can be a potential therapeutic agent for wound healing.

Magnetic coupling modulation in meta-nitroxide-functionalized isoalloxazine magnets with redox-active units as efficient side-modulators.

Magnetic conversion can be accomplished in a variety of ways, as organic molecules with switchable magnetic characteristics offer numerous technological applications. It is crucial to find magnetism-switchable systems because, in the field of organic magnetic materials, the redox-induced magnetic reversal is very simple to achieve and shows significant applications. Herein, we computationally design isoalloxazine-based diradicals through oxidizing N10 and adding a nitroxide to C8 as the spin source (i.e. 8-nitroxide-isoalloxazine 10-oxide, an m-phenylene-like nitroxide diradical expanded with a redox unit as a side-modulator) and its N1/N5-hydrogenated/protonated diradical derivatives and introducing substituents (-OH, -NH2, and -NO2) to C6. We demonstrate that the basically modified structure exhibits ferromagnetic (FM) characteristics with a magnetic coupling constant (J) of 561.3 cm-1 calculated at the B3LYP/6-311+G(d,p) level, obeying the meta-phenylene-mediated diradical character, and dihydrogenation can lead to an AFM diradical with considerably large J (-976.1 cm-1). Surprisingly, protonation at N1 or N5 can lead to distinctly different magnetic variations (561.3 → -1602.9 cm-1 at N1 versus 561.3 → 379.1 cm-1 at N5). Analyses indicate that small singlet-triplet energy gaps and small energy gaps between the highest occupied and lowest unoccupied molecular orbitals (HOMO, LUMO) of the closed shell singlet state are the key features of these isoalloxazine diradicals, and aromaticity variations, significant spin delocalization from the π-conjugated structure and spin polarization from the non-Kekule structure induced by modification are responsible for the magnetic conversion. Furthermore, the spin alternation rule, the singly occupied molecular orbital (SOMO) effect, and the SOMO-SOMO energy splitting of the triplet state are used to analyze these distinct variations. This work provides a novel understanding of the structures and characteristics of modified isoalloxazine diradicals, as well as essential details for the intricate design and characterization of new isoalloxazine-based potential organic magnetic switches.

Intramolecular Proton Transfer Modulation of Magnetic Spin Coupling Interaction in Photochromic Azobenzene Derivatives with an Ortho-Site Hydroxyl as a Modulator.

Proton transfer modulation in an organic diradical is apparently the most conspicuously attractive phenomenon. In this work, we have computationally designed the trans and cis forms of photochromic azobenzene- (AB-) bridged diradicals by considering AB as coupler and two nitroxide (NO) as spin sources and a -OH attaching at the ortho site as modulator. Our object is that through intramolecular proton transfer to protonate the azo-unit (-N═N-) the magnetic coupling characteristics of the designed diradicals can be modulated in their photocontrolled trans and cis forms. The calculated results indicate that PT can significantly regulate the magnetic spin coupling constants, J = -701.3 cm-1 ↔ -286.2 cm-1 for the trans form and -544.1 cm-1 ↔ -328.1 cm-1 for the cis form. In particular, we discover that these designed magnetic molecules can undergo magnetic conversion between antiferromagnetic and ferromagnetic coupling through PT, besides there is considerable increase in the magnitude of their magnetic coupling constants J, (e.g., -59.97 to 172.4 cm-1) for the trans-mode at the m/m linking site. Moreover, we discover that the nitroxide radicals at different linking positions have a significant impact and remarkably alter the magnetic spin coupling characteristics of AB-based diradicals. Besides, various radical groups are used as spin sources which corroborated our assumptions and tended to the same conclusion. This work offers a novel understanding of the spin interaction mechanism and a viable approach for the rational design of new AB-based magnets which are beneficial for further applications in the future.

The Drudgery of a Doctor's Disciple: Exploring the effects of Negative Role Modelling on medical students' professional development.

INTRODUCTION: Role modelling is considered an essential component of teaching in various educational settings and impact students' professional development. Most studies found in the literature have explored the influence of positive role models and limited data is available about the negative role modelling. This study explores the effects of negative role modelling of teachers on the professional development of future doctors. METHODS: A qualitative exploratory study was done in three medical colleges of Lahore, Pakistan. Fifteen, telephonic, semi-structured interviews were done with fifth-year MBBS students. A thematic analysis was done through manual coding of transcribed interviews. RESULTS: A total of 374 codes were generated in the first cycle of coding, that was merged to 42 in the second cycle. These codes led to 4 subthemes that finally emerged as two themes. The first theme was "Students & patients: In the same boat" highlighting the damaging effects of negative role modelling. The second theme was "Taking the bad with the good" focusing on the ambivalent response of students towards the unprofessional behaviours of their role models. CONCLUSION: Negative role modelling exponentially affects the attitude and behaviour of medical students especially in informal settings and have detrimental effects on patient care. Students lose some degree of humanism while unconsciously observing the unethical behaviours, to become a part of hospital culture whereas some students show determination to channelize their resentment to reforms.

Pediatric Residency Preparedness for Pediatric Emergency Medicine Fellowship.

OBJECTIVE: The aim of the study was to examine the perception of pediatric emergency medicine (PEM) program directors (PDs), associate PDs (APDs) and first-year fellows regarding the preparedness of new PEM fellows who have graduated from pediatric residency programs. METHODS: Unique surveys were created and electronically distributed to PEM PDs/APDs and first-year PEM fellows. Individual and institutional demographic information was collected. Using a 5-point Likert scale, survey items centered the perceived preparedness of first-year PEM fellows within 5 domains: professionalism, independence/autonomy, psychomotor skills, clinical evaluation and management, and academia/scholarship. RESULTS: Forty percent (48/119) of eligible PDs and APDs and 56% (82/147) of eligible first-year PEM fellows responded. Most PDs/APDs strongly agreed or agreed that incoming fellows perform adequately in areas of professionalism, independence/autonomy, and clinical evaluation and management. The PDs/APDs perceived fellows to be less prepared in the academia/scholarship domain and several psychomotor skills. Most first-year PEM fellows perceived themselves as prepared in areas of professionalism and clinical evaluation and management. Fellows had varied feelings of preparedness in the domains of independence/autonomy, psychomotor skills, and academia/scholarship. Overall, most PDs/APDs (54%) and fellows (84%) feel that pediatric residency training was strong or very strong. CONCLUSIONS: Most respondents in both groups felt that in general, pediatric residency programs adequately train residents for PEM fellowship. Both groups felt that the strengths of general pediatric training were among the domains of professionalism and clinical evaluation and management, whereas psychomotor skills and academia and scholarship were areas of improvement. These findings may be used by general pediatric residency and PEM fellowship programs to guide curriculum development.

Point-of-Care Ultrasound to Evaluate the Acute Abdomen: A Case of Bowel Perforation After Unknown Single Magnet Ingestion.

ABSTRACT: We report the case of a 3-year-old boy who presented to the pediatric emergency department in undifferentiated shock with an acute abdomen. Point-of-care ultrasound revealed viscous perforation with a large amount of free fluid. Intraoperatively, a single magnet was discovered as the likely cause of bowel perforation and the resulting state of shock.

Agreement With Pediatric Suprapatellar Bursa Effusion Assessments by Point-of-Care Ultrasound After Remote Training.

BACKGROUND: The ease of instruction for point-of-care ultrasound (POCUS) to detect suprapatellar bursa (SPB) effusions in pediatric patients is unknown. Considering in person limitations because of the coronavirus pandemic, strategies for POCUS education by remote learning are necessary. METHODS: We crafted a 90-minute didactic training that was presented via a remote learning format. The main outcome of interest was the interobserver reliability of SPB effusion assessment by novice sonologists compared with POCUS faculty. Novice sonologists were pediatric emergency medicine (PEM) fellows. Pediatric emergency medicine fellows interpreted longitudinal SPB examinations obtained in our pediatric emergency department from July 2013 to June 2020. Assessments were performed 2 months after the remote training. Pediatric emergency medicine fellows had a limited experience performing these musculoskeletal scans and were blinded to POCUS faculty and each other's assessments. Interobserver reliability was assessed with Cohen κ coefficient. Second, we calculated test characteristics of knee radiography compared with PEM POCUS faculty determination of SPB effusion by ultrasound. We further explored how effusion size measured by POCUS impacted the diagnosis by knee radiography. A receiver operator characteristic curve of knee radiography diagnosis of SPB effusion was created using the maximal height of SPB effusion by POCUS as the predictor variable. RESULTS: A total of 116 SPB scans in 71 patients were assessed. From this group, 70 scans were of affected knees and 46 scans were of contralateral, asymptomatic knees. The mean age of patients was 10 years and 46% were girl. The prevalence of SPB effusions was 42%. The κ coefficients between the 3 novice sonologists and POCUS faculty were 0.75 (0.62-0.87), 0.77 (0.65-0.89), and 0.83 (0.72-0.93) with 88%, 89%, and 91% agreement. Knee radiography exhibited an overall sensitivity of 65% (95% confidence interval [CI], 46-79%), specificity of 84% (95% CI, 60-97%), negative predictive value of 55% (95% CI, 43-66%), and positive predictive value of 88% (95% CI, 73-96%) to diagnose SPB effusions. The area under the receiver operator characteristic curve was 0.850. With an SPB height cutoff of 4 mm as true positives, radiography had a sensitivity of 81% and a specificity of 83%. CONCLUSIONS: After a remote teleconference didactic session, PEM fellows were able to successfully diagnose SPB effusions using a longitudinal view with substantial interobserver reliability. Knee radiography exhibited limited sensitivity to rule out SPB effusions.

A Workplace Procedure Training Cart to Augment Pediatric Resident Procedural Learning.

OBJECTIVE: Our primary aim was to describe pediatric residents' use of a workplace procedural training cart. An exploratory aim was to examine if the cart associated with increased resident procedural experiences with real patients. METHODS: Guided by the procedural training construct of "Learn, See, Practice, Prove, Do, Maintain," we created a novel workplace procedural training cart with videos (learn and see) and simulation equipment (practice and prove). An electronic logbook recorded resident use data, and a brief survey solicited residents' perceptions of the cart's educational impact. We queried our electronic medical record to compare the proportion of real procedures completed by residents before and after the intervention. RESULTS: From August 1 to December 31, 2019, 24 pediatric residents (10 interns and 14 seniors) rotated in the pediatric emergency department. Twenty-one cart encounters were logged, mostly by interns (67% [14/21]). The 21 cart encounters yielded 32 learning activities (8 videos watched and 24 procedures practiced), reflecting the residents' interest in laceration repair (50% [4/8], 54% [13/24]) and lumbar puncture (38% [3/8], 33% [8/24]). All users agreed (29% [6/21]) or strongly agreed (71% [15/21]) the cart encouraged practice and improved confidence in independently performing procedures. No changes were observed in the proportion of actual procedures completed by residents. CONCLUSIONS: A workplace procedural training cart was used mostly by pediatric interns. The cart cultivated residents' perceived confidence in real procedures but was not used by all residents or influenced residents' procedural behaviors in the pediatric emergency department.

POCUS Case Review: Considering Superior Mesenteric Artery Syndrome in the Pediatric Emergency Department.

ABSTRACT: We report a case of a 16-year-old adolescent girl who presented to the pediatric emergency department for worsening abdominal pain and vomiting and had significant weight loss over the previous 4 months. Point-of-care ultrasound was used to assess for signs of superior mesenteric artery syndrome.

Evaluation of Hip Pain and Management of Toxic Synovitis in the Ultrasound Era.

ABSTRACT: The cause of acute onset hip pain in children can be difficult to determine. Once trauma is excluded, the workup revolves around determining whether there is a hip effusion and eliminating orthopedic emergencies. Point-of-care-ultrasound can be used as an adjunct in the workup. In this article, we review (1) differential diagnosis of hip pain, with a focus on toxic synovitis; (2) the evaluation of a hip for the presence of effusion, including the point-of-care ultrasound technique; and (3) the management of toxic synovitis.

The impact of caregiver health literacy on healthcare outcomes for low income minority children with asthma.

Objectives: The aim of this exploratory study was to assess the impact of caregiver health literacy (HL) on health care outcomes for their child with asthma.Methods: Caregiver dyads across two different healthcare delivery systems completed a battery of validated asthma outcome instruments, including the Newest Vital Sign™ as a measure of HL for the caregivers of children ages 7-18 y. Utilization history was obtained through the electronic medical record. Descriptive analysis with bivariate associations was conducted.Results: There was no direct relationship between HL and asthma outcomes in the 34 Hispanic and African American caregiver-child dyads. However, caregiver health literacy was significantly related to language (p = 0.02). African American English-speaking caregivers, seen in an urban emergency department, demonstrated adequate health literacy. Hispanic Spanish-speaking caregivers, seeking care in a mobile asthma van, showed limited health literacy. There was no significant association between caregivers' HL and routine asthma care visits when language and child age were controlled.Conclusions: Assessing patient factors can identify persons at risk who need additional support to negotiate the healthcare system when providing care for a child with asthma.

Priming GPCR signaling through the synergistic effect of two G proteins.

Although individual G-protein-coupled receptors (GPCRs) are known to activate one or more G proteins, the GPCR-G-protein interaction is viewed as a bimolecular event involving the formation of a ternary ligand-GPCR-G-protein complex. Here, we present evidence that individual GPCR-G-protein interactions can reinforce each other to enhance signaling through canonical downstream second messengers, a phenomenon we term "GPCR priming." Specifically, we find that the presence of noncognate Gq protein enhances cAMP stimulated by two Gs-coupled receptors, ß2-adrenergic receptor (ß2-AR) and D1 dopamine receptor (D1-R). Reciprocally, Gs enhances IP1 through vasopressin receptor (V1A-R) but not α1 adrenergic receptor (α1-AR), suggesting that GPCR priming is a receptor-specific phenomenon. The C terminus of either the Gαs or Gαq subunit is sufficient to enhance Gα subunit activation and cAMP levels. Interaction of Gαs or Gαq C termini with the GPCR increases signaling potency, suggesting an altered GPCR conformation as the underlying basis for GPCR priming. We propose three parallel mechanisms involving (i) sequential G-protein interactions at the cognate site, (ii) G-protein interactions at distinct allosteric and cognate sites on the GPCR, and (iii) asymmetric GPCR dimers. GPCR priming suggests another layer of regulation in the classic GPCR ternary-complex model, with broad implications for the multiplicity inherent in signaling networks.

Structural Elements in the Gαs and Gαq C Termini That Mediate Selective G Protein-coupled Receptor (GPCR) Signaling.

Although the importance of the C terminus of the α subunit of the heterotrimeric G protein in G protein-coupled receptor (GPCR)-G protein pairing is well established, the structural basis of selective interactions remains unknown. Here, we combine live cell FRET-based measurements and molecular dynamics simulations of the interaction between the GPCR and a peptide derived from the C terminus of the Gα subunit (Gα peptide) to dissect the molecular mechanisms of G protein selectivity. We observe a direct link between Gα peptide binding and stabilization of the GPCR conformational ensemble. We find that cognate and non-cognate Gα peptides show deep and shallow binding, respectively, and in distinct orientations within the GPCR. Binding of the cognate Gα peptide stabilizes the agonist-bound GPCR conformational ensemble resulting in favorable binding energy and lower flexibility of the agonist-GPCR pair. We identify three hot spot residues (Gαs/Gαq-Gln-384/Leu-349, Gln-390/Glu-355, and Glu-392/Asn-357) that contribute to selective interactions between the ß2-adrenergic receptor (ß2-AR)-Gαs and V1A receptor (V1AR)-Gαq The Gαs and Gαq peptides adopt different orientations in ß2-AR and V1AR, respectively. The ß2-AR/Gαs peptide interface is dominated by electrostatic interactions, whereas the V1AR/Gαq peptide interactions are predominantly hydrophobic. Interestingly, our study reveals a role for both favorable and unfavorable interactions in G protein selection. Residue Glu-355 in Gαq prevents this peptide from interacting strongly with ß2-AR. Mutagenesis to the Gαs counterpart (E355Q) imparts a cognate-like interaction. Overall, our study highlights the synergy in molecular dynamics and FRET-based approaches to dissect the structural basis of selective G protein interactions.

Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins.

G protein α subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of Gαi. Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function.

Synthesis of Some Unique Carbamate Derivatives bearing 2-Furoyl-1-piperazine as a Valuable Therapeutic Agents.

The aim of the research work was to synthesize different biologically active carbamate derivatives bearing 2-furoyl-1-piperazine and having modest toxicity. The synthesis was completed as a multiple sequence. The structural confirmation of all the synthesized compounds was obtained by EI-MS, IR and 1H-NMR spectral data. The enzyme inhibition and antibacterial potential of the synthesized compounds was evaluated. To find the utility of the prepared compounds as possible therapeutic agents their cytotoxicity was also checked. All the compounds were active against acetylcholinesterase enzyme, especially 12 and 14 showed very good inhibitory potential relative to Eserine, a reference standard. Almost all the compounds showed good activities against both Gram-positive and Gram-negative bacterial strains.

Predictive accuracy of anti mullerian hormone as indicator of ovarian follicle loss in cyclophosphamide treated mice.

OBJECTIVE: To evaluate the strength of anti-mullerian hormone in reflecting the stages of ovarian toxicity-induced by cyclophosphamide. METHODS: This study was conducted in December 2014 and comprised female mice that were divided into four groups: group A served as control, group B received three weekly injections of cyclophosphamide, group C was co administered alpha-tocopherol along with cyclophosphamide, while group D solely received alpha-tocopherol. The ovaries were evaluated for follicular dynamics, and anti-mullerian hormone was assessed using mouse enzyme-linked immunosorbent assay kit. The data was analysed using SPSS 19. RESULTS: There were 40 mice in the study. Histological analysis revealed severely reduced ovarian reserve in group B(p<0.01).In group C alpha-tocopherol conserved the ovarian reserve to near normal, thus follicle count was significantly higher than group B (p<0.05). However, this moderate reduction was still lower than the controls (p<0.01). Furthermore, the number of corpus lutea and atretic follicles were significantly higher in groups B and C (p<0.01). Regarding hormonal analyses in comparison to controls, anti-mullerian hormone levels were low in group B (p<0.01), while group C reported an insignificant fall in serum anti-mullerian hormone levels (p=0.101). CONCLUSIONS: There was substantial evidence that anti-mullerian hormone monitoring during chemotherapy administration may fulfil the criteria of earliest diagnostic indicator of secondary infertility.

Pheromone sensing regulates Caenorhabditis elegans lifespan and stress resistance via the deacetylase SIR-2.1.

Lifespan in Caenorhabditis elegans, Drosophila, and mice is regulated by conserved signaling networks, including the insulin/insulin-like growth factor 1 (IGF-1) signaling cascade and pathways depending on sirtuins, a family of NAD(+)-dependent deacetylases. Small molecules such as resveratrol are of great interest because they increase lifespan in many species in a sirtuin-dependent manner. However, no endogenous small molecules that regulate lifespan via sirtuins have been identified, and the mechanisms underlying sirtuin-dependent longevity are not well understood. Here, we show that in C. elegans, two endogenously produced small molecules, the dauer-inducing ascarosides ascr#2 and ascr#3, regulate lifespan and stress resistance through chemosensory pathways and the sirtuin SIR-2.1. Ascarosides extend adult lifespan and stress resistance without reducing fecundity or feeding rate, and these effects are reduced or abolished when nutrients are restricted. We found that ascaroside-mediated longevity is fully abolished by loss of SIR-2.1 and that the effect of ascr#2 requires expression of the G protein-coupled receptor DAF-37 in specific chemosensory neurons. In contrast to many other lifespan-modulating factors, ascaroside-mediated lifespan increases do not require insulin signaling via the FOXO homolog DAF-16 or the insulin/IGF-1-receptor homolog DAF-2. Our study demonstrates that C. elegans produces specific small molecules to control adult lifespan in a sirtuin-dependent manner, supporting the hypothesis that endogenous regulation of metazoan lifespan functions, in part, via sirtuins. These findings strengthen the link between chemosensory inputs and conserved mechanisms of lifespan regulation in metazoans and suggest a model for communal lifespan regulation in C. elegans.

Serum anti-mullerian hormone: Correlation with the ovarian follicular dynamics in healthy mice.

OBJECTIVE: To evaluate the relationship between serum anti-Mullerian hormone and follicular dynamics in mice. METHODS: This experimental study was conducted in November, 2014 at the Dow University of Health Sciences, Karachi, and comprised laboratory-bred albino mice. They were sacrifised under anaesthesia and blood was collected via cardiac puncture to assess anti-Mullerian hormone while ovaries were collected for morphometric analyses. SPSS 19 was used for data analysis. RESULTS: There were 20 mice with a mean weight of 25±1.89 grams, while weight of the ovaries obtained from these mice was 9.6±0.92mg. The mean serum anti-Mullerian hormone was 29.89±9.7ng/ml. On average, there were 87.8+13.54 primordial follicles, 51.85±8.36 primary, 20.35±5.57 secondary, 11.30±3.38 early antral and 3.05 ± 1.27 late antral follciles (p<0.001; p=0.06).. CONCLUSIONS: Association of anti-Mullerian hormone with follicle dynamics reflected its role as a true ovarian reserve marker. Its assessment was of great significance in infertile women as well as young patients receiving chemotherapy.

Focal amyotrophy in multiple sclerosis.

INTRODUCTION: The assumption that multiple sclerosis (MS) is purely a white matter disease has been challenged in recent years by observations of axonal damage and neuronal loss in gray matter of the cortex, subcortex, and spinal cord. METHODS: We report the case of a 71-year-old man with primary progressive MS and longstanding right arm weakness who presented with intermittent right arm pain. RESULTS: Neurological examination showed atrophy, weakness, and hyporeflexia, and electromyography (EMG) showed acute and chronic partial denervation in multiple segments of the right arm. Magnetic resonance imaging (MRI) demonstrated asymmetric volume loss and increased T2 signal in the right anterior spinal cord from C3 to C7, with no evidence of nerve root compression. CONCLUSIONS: Lower motor neuron involvement of his right arm was caused by MS with involvement of either the anterior horn cells or the intraspinal motor nerve roots.

Detection of G protein-selective G protein-coupled receptor (GPCR) conformations in live cells.

Although several recent studies have reported that GPCRs adopt multiple conformations, it remains unclear how subtle conformational changes are translated into divergent downstream responses. In this study, we report on a novel class of FRET-based sensors that can detect the ligand/mutagenic stabilization of GPCR conformations that promote interactions with G proteins in live cells. These sensors rely on the well characterized interaction between a GPCR and the C terminus of a Gα subunit. We use these sensors to elucidate the influence of the highly conserved (E/D)RY motif on GPCR conformation. Specifically, Glu/Asp but not Arg mutants of the (E/D)RY motif are known to enhance basal GPCR signaling. Hence, it is unclear whether ionic interactions formed by the (E/D)RY motif (ionic lock) are necessary to stabilize basal GPCR states. We find that mutagenesis of the ß2-AR (E/D)RY ionic lock enhances interaction with Gs. However, only Glu/Asp but not Arg mutants increase G protein activation. In contrast, mutagenesis of the opsin (E/D)RY ionic lock does not alter its interaction with transducin. Instead, opsin-specific ionic interactions centered on residue Lys-296 are both necessary and sufficient to promote interactions with transducin. Effective suppression of ß2-AR basal activity by inverse agonist ICI 118,551 requires ionic interactions formed by the (E/D)RY motif. In contrast, the inverse agonist metoprolol suppresses interactions with Gs and promotes Gi binding, with concomitant pertussis toxin-sensitive inhibition of adenylyl cyclase activity. Taken together, these studies validate the use of the new FRET sensors while revealing distinct structural mechanisms for ligand-dependent GPCR function.