Exploring GPCR conformational dynamics using single-molecule fluorescence.

G protein-coupled receptors (GPCRs) are membrane proteins that transmit specific external stimuli into cells by changing their conformation. This conformational change allows them to couple and activate G-proteins to initiate signal transduction. A critical challenge in studying and inferring these structural dynamics arises from the complexity of the cellular environment, including the presence of various endogenous factors. Due to the recent advances in cell-expression systems, membrane-protein purification techniques, and labeling approaches, it is now possible to study the structural dynamics of GPCRs at a single-molecule level both in vitro and in live cells. In this review, we discuss state-of-the-art techniques and strategies for expressing, purifying, and labeling GPCRs in the context of single-molecule research. We also highlight four recent studies that demonstrate the applications of single-molecule microscopy in revealing the dynamics of GPCRs. These techniques are also useful as complementary methods to verify the results obtained from other structural biology tools like cryo-electron microscopy and x-ray crystallography.

Single-molecule visualization of human A2A adenosine receptor activation by a G protein and constitutively activating mutations.

Mutations that constitutively activate G protein-coupled receptors (GPCRs), known as constitutively activating mutations (CAMs), modify cell signaling and interfere with drugs, resulting in diseases with limited treatment options. We utilize fluorescence imaging at the single-molecule level to visualize the dynamic process of CAM-mediated activation of the human A2A adenosine receptor (A2AAR) in real time. We observe an active-state population for all CAMs without agonist stimulation. Importantly, activating mutations significantly increase the population of an intermediate state crucial for receptor activation, notably distinct from the addition of a partner G protein. Activation kinetics show that while CAMs increase the frequency of transitions to the intermediate state, mutations altering sodium sensitivity increase transitions away from it. These findings indicate changes in GPCR function caused by mutations may be predicted based on whether they favor or disfavor formation of an intermediate state, providing a framework for designing receptors with altered functions or therapies that target intermediate states.

Maternal and Neonatal Outcomes in Women with Metabolic Syndrome and Substance Use Disorder.

INTRODUCTION: Metabolic syndrome amplifies the risk of gestational diabetes, preeclampsia, and preterm labor in pregnant women. Similarly, women with substance use disorder have worsened obstetric and birth outcomes. Despite these two conditions being major healthcare disparities in Appalachia, the health outcomes of this cohort have not been studied thus far. This study looks at the health outcomes of this cohort. METHOD AND RESULTS: In this retrospective cohort study, we analyzed 27,955 mothers who delivered at Cabell Huntington Hospital between January 2010 and November 2021. We implemented Chi-square tests to determine the associations and multiple logistic regression methods for comparison after controlling for other factors, and found that MetS, together with SUD, significantly increases the risk as well as the number of pregnancy complications such as gestational diabetes (p-value < 0.001), preeclampsia (p-value < 0.001), premature rupture (p-value < 0.001), preterm labor (p-value < 0.001), and newborn disorder (p-value < 0.001) compared to the women who had none or had either MetS or SUD alone. CONCLUSION: Women with both metabolic syndrome and substance abuse had worsened pregnancy and neonatal outcomes compared to women with metabolic syndrome or SUD alone. In conclusion, analysis of all the variables is crucial to strategically planning and implementing health interventions that will positively influence the health outcome of the pregnant woman as well as the child.

Single-molecule analysis reveals that a glucagon-bound extracellular domain of the glucagon receptor is dynamic.

Dynamic information is vital to understanding the activation mechanism of G protein-coupled receptors (GPCRs). Despite the availability of high-resolution structures of different conformational states, the dynamics of those states at the molecular level are poorly understood. Here, we used total internal reflection fluorescence microscopy to study the extracellular domain (ECD) of the glucagon receptor (GCGR), a class B family GPCR that controls glucose homeostasis. Single-molecule fluorescence resonance energy transfer was used to observe the ECD dynamics of GCGR molecules expressed and purified from mammalian cells. We observed that for apo-GCGR, the ECD is dynamic and spent time predominantly in a closed conformation. In the presence of glucagon, the ECD is wide open and also shows more dynamic behavior than apo-GCGR, a finding that was not previously reported. These results suggest that both apo-GCGR and glucagon-bound GCGRs show reversible opening and closing of the ECD with respect to the seven-transmembrane (7TM) domain. This work demonstrates a molecular approach to visualizing the dynamics of the GCGR ECD and provides a foundation for understanding the conformational changes underlying GPCR activation, which is critical in the development of new therapeutics.

Aspergillus Coinfection in a Hydatid Cyst Cavity of Lung in an Immunocompetent Host: A Case Report and Review of Literature.

Aspergilloma (a saprophytic infection) typically colonizes lung cavities due to underlying diseases such as tuberculosis, bronchiectasis, cavitary lung cancer, sarcoidosis, and pulmonary infarctions. Rarely, aspergilloma has been noted within a hydatid cyst. Even if this was the case, it is more common to find the coexistence of aspergilloma and pulmonary echinococcal cysts in immunocompromised individuals. It is, however, very uncommon to find this coinfection in normal immune status individuals. Here, we report on the successfully treated case of a 30-year-old immunocompetent female from Western Nepal with histologically proven coinfection by these two pathogens. She had a prolonged history of exposure to domesticated dogs. She suffered from hemoptysis from time to time for 3 years with increased frequency in the last 30 days. She was misdiagnosed clinically during a past medical visit at a local health center. Her computed tomography (CT) scans showed well-defined nonenhancing cystic lesions in the anterior basal segment of the right lower lobe adjacent to the major fissure. She underwent enucleation of the cyst via right posterolateral thoracotomy. On further histopathological evaluation, laminated membranes of the ectocyst along with fungal elements were found, and periodic acid-Schiff (PAS) staining revealed Aspergillus in the form of septate hyphae and acute angle branching. Owing to patient's economic constraints and unavailability in our center, DNA testing and molecular characterization could not be performed which further highlights the essence of diagnosing and managing such cases in resource poor settings. Eventually, we reviewed 12 confirmed cases of this coinfection in immunocompetent individuals during a period of 7 years (2015-2022) comparing them to a systematic review of 22 confirmed cases of the same coinfection from 1995 to 2014.

Combined personalized therapy for the treatment of multiple giant keloids: a case report and literature review.

Keloids are the result of an abnormal wound-healing process and are associated with various risk factors. The majority of diagnoses are clinical. Successful treatment of keloid is challenging due to its nonregressing and recurring nature. Case presentation: We discuss the case of a 30-year-old mongoloid male who had multiple swellings over his body for the past 10 years. More striking are the giant keloids that are present over his bilateral scapulae. Diagnosis of keloid was made clinically. Smaller sessile lesions over his shoulder and upper limbs were subjected to intralesional 5-fluorouracil and triamcinolone injections, whereas the giant bilateral scapular keloids underwent excision and split skin grafting. Clinical discussion: Keloids usually present with firm and rubbery masses that extend beyond the site of the previous wound/injury. Keloids are diagnosed and evaluated clinically. Its differentiation from the hypertrophic scar is done based on the presence of multiple lesions beyond the site of the previous wound/injury. Conclusion: Treatment of keloids is difficult due to their nonregressing and recurring nature. Hence, the main goal of treatment is to tailor the therapy to the patient's needs such that the benefits outweigh the risks.

Prenatal Perception of WIC Breastfeeding Recommendations Predicts Breastfeeding Exclusivity and Duration in the Infants' First Year.

BACKGROUND: Pregnant participants who perceived that the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) recommends breastfeeding only were more likely to have better early breastfeeding outcomes. OBJECTIVES: Our objective was to examine the association between prenatal perception of WIC's breastfeeding recommendations and breastfeeding duration through the first year of infant life. METHODS: This observational study used a national longitudinal sample of 1594 pregnant participants in the WIC Infant and Toddler Feeding Practices Study-2 in 2013. Four measures of breastfeeding duration were used: 1) a discrete measure of exclusive breastfeeding through 5 mo; 2) a continuous measure of exclusive breastfeeding (in days up to 7 mo); 3) a discrete measure of any breastfeeding through 11 mo; and 4) a continuous measure of any breastfeeding (in days up to 13 mo). The primary explanatory variable was the participant's prenatal perception of whether WIC recommended breastfeeding only. The univariate analyses of time to breastfeeding cessation were performed using Kaplan-Meier curves. The Cox regression model was adopted to estimate the likelihood of breastfeeding outcomes over time. All analyses accounted for complex survey design effects. RESULTS: Compared with their peers who perceived WIC to recommend formula only or both breastfeeding and formula equally, participants who perceived WIC as recommending breastfeeding only were less likely to stop exclusive breastfeeding through 5 mo (HR = 0.83; 95% CI: 0.69, 0.99) or to stop any breastfeeding through 11 mo (HR = 0.80; 95% CI: 0.69, 0.92), without controlling for prenatal infant feeding intentions. Similar patterns were observed in the 2 continuous measures, as they were also less likely to stop exclusive breastfeeding by 7 mo (HR = 0.78; 95% CI: 0.69, 0.90) or to stop any breastfeeding by 13 mo (HR = 0.82; 95% CI: 0.71, 0.95). CONCLUSIONS: Prenatal perception of WIC's breastfeeding recommendation can be a useful predictor of breastfeeding duration in WIC participants.

Safety and efficacy of endovascular thrombectomy in patients with severe cerebral venous thrombosis: A meta-analysis.

Background: Cerebral venous thrombosis (CVT) is a rare thrombotic condition which is traditionally treated with anti-coagulation therapy. Subsets of patients with severe CVT have been treated with endovascular thrombectomy (EVT). Despite the high estimated mortality associated with severe CVT, there has been only one randomized control trial done regarding safety and efficacy of EVT in severe CVT compared to standard medical management. Evidence in this area is lacking. Objective: The aim of this systematic review is to analyze all existing literature and generate robust information regarding the role of EVT in the management of patients with severe CVT. Methods: This systematic review and meta-analysis followed PRISMA guideline. PubMed, Embase, Google Scholar, and CNKI were searched for eligible studies from 2007 to 2021. Safety and efficacy of EVT were evaluated by meta-analyzing recanalization status, the good functional outcome at follow-up, recurrent CVT, new hematoma. A pooled proportion with a 95% confidence interval was derived from a meta-analysis of various outcomes (CI). Results: A total of 33 studies comprising 610 patients treated with EVT were included for analysis which comprised one randomized control trial, one prospective study and 31 retrospective studies. Based on pooled data, 85% of patients had good functional outcome, 62% had complete recanalization, 5% had all-cause mortality, and 3% had catheter related complications. The efficacy outcomes in this analysis had a significant heterogeneity and a subgroup analysis was also done to explain these findings. The minimum time of follow up was 3 months and varied EVT techniques were used across the studies. Conclusion: This meta-analysis suggests EVT may be safe and efficacious in treating patients with severe CVT. Registration: Our protocol was registered with PROSPERO: International prospective register of systematic reviews with the registration number CRD42021254760.

Cleidocranial dysplasia-A case report of incidentally found and lately diagnosed disorder.

Cleidocranial dysplasia (CCD) is a rare autosomal dominant disorder with facial, dental, and skeletal impairments. Affected individuals have varying degrees of skull, shoulder, dental, spine, and facial impairments. Early diagnosis and timely intervention help in minimization of complications, planning of pregnancy, and better quality of life.

Intake assessments of salivary cortisol, survey responses, and adverse childhood experiences are associated with recovery success in an abstinence-based treatment program for substance use disorders.

BACKGROUND: Connecting patients to treatment for a substance use disorder (SUD) that satisfies their needs is often complicated by confounding factors. A reliable measurement of patients' underlying stress level may be helpful because it often reflects many of the same confounders as their SUD. Whereas cortisol levels reflect physiological responses to stress, patients' cortisol levels during recovery from an SUD may serve as biomarkers for stressors that result in poor treatment outcomes, including early discontinuation of treatment. However, further exploration of the relationship between patients' cortisol levels and their treatment outcomes is needed for this approach to be clinically useful. METHODS: We enrolled participants from an abstinence-based, male-only, residential alcohol and drug recovery program to examine the relationship between salivary cortisol, stress exposure, ACEs, and treatment retention. RESULTS: Participants who remained in the program <90 days had significantly higher initial cortisol levels than those who remained ≥90 days (0.62 ± 0.074 µg/dl vs. 0.36 ± 0.037 µg/dl). Kaplan-Meier curves differed significantly when we grouped participants according to whether their cortisol level was below or above the overall average of 0.49 ± 0.044 µg/dl, with the median numbers of days before discontinuing being 110 and 60, respectively. A Cox proportional hazards model indicated that elevated salivary cortisol (with increases in µg/dl), marital/relationship status, and adverse childhood experiences (ACEs) score correlated significantly with hazards of discontinuing the program (hazard ratios for the three factors were 3.49, 2.39, and 1.50, respectively). DISCUSSION: Cortisol level may predict, at least partially, SUD treatment program retention regardless of individuals' numerous confounding factors or the substance used. If this approach is validated, it could enable providers to consider patients' cortisol levels at the time of admission to treatment to facilitate their retention in treatment and thereby enhance their recovery.

Production of human A2AAR in lipid nanodiscs for 19F-NMR and single-molecule fluorescence spectroscopy.

We describe production of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor (GPCR) for 19F-NMR and single-molecule fluorescence (SMF) spectroscopy. We explain in detail steps shared between the two sample preparation strategies, including expression and isolation of A2AAR and assembly of A2AAR in lipid nanodiscs and procedures for incorporation of either 19F-NMR or fluorescence probes. Protocols for SMF experiments include sample setup, data acquisition, data processing, and error analysis. For complete details on the use and execution of this protocol, please refer to Wei et al. (2022) and Susac et al. (2018).

Slow conformational dynamics of the human A2A adenosine receptor are temporally ordered.

A more complete depiction of protein energy landscapes includes the identification of different function-related conformational states and the determination of the pathways connecting them. We used total internal reflection fluorescence (TIRF) imaging to investigate the conformational dynamics of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor (GPCR), at the single-molecule level. Slow, reversible conformational exchange was observed among three different fluorescence emission states populated for agonist-bound A2AAR. Transitions among these states predominantly occurred in a specific order, and exchange between inactive and active-like conformations proceeded through an intermediate state. Models derived from molecular dynamics simulations with available A2AAR structures rationalized the relative fluorescence emission intensities for the highest and lowest emission states but not the transition state. This suggests that the functionally critical intermediate state required to achieve activation is not currently visualized among available A2AAR structures.

EDTA-dependent pseudo thrombocytopenia mimicking dengue fever-associated persistent thrombocytopenia: A case report.

No hemorrhagic manifestations, presence of platelet clumps on the peripheral blood smear, normal manual count, and normal autoanalyzer count after collecting blood in citrate vial help confirm the diagnosis of EDTA-dependent thrombocytopenia.

Prenatal perception of breastfeeding recommendations predicts early breastfeeding outcomes of participants in the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC).

BACKGROUND: Prenatal psychosocial factors predict breastfeeding practices but are not assessed in the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC). OBJECTIVES: This study examined how prenatal perceptions of WIC's breastfeeding recommendations were associated with early breastfeeding outcomes. METHODS: This study used longitudinal data from a national sample of 2053 pregnant participants in the WIC Infant and Toddler Feeding Practices Study-2 (WIC ITFPS-2) in 2013, the only national data assessing prenatal perceptions of WIC's breastfeeding recommendations. Early breastfeeding outcomes included breastfeeding initiation, breast milk first fed after birth, breastfeeding in the first hour, breast milk first fed after leaving the hospital, and breastfeeding status at the first and third months. The primary predictor was the participant's prenatal perception of whether WIC recommended breastfeeding only or not. Log-binomial regression was used with adjustment for socio-demographics, previous breastfeeding, WIC participation, breastfeeding support, and infant feeding intentions (IFI). RESULTS: Without controlling for IFI, the perception of WIC recommending breastfeeding only predicted breastfeeding outcomes positively. The risk ratio (RR) associated with prenatal perceptions varied from 1.14 (95% CI: 1.03, 1.25; P = 0.008) for breastfeeding in the first hour, to 1.27 (95% CI: 1.12, 1.43; P < 0.001) for breast milk first fed after leaving hospital, to 1.66 (95% CI: 1.35, 2.04; P < 0.001) for 3-mo breastfeeding only. After controlling for IFI, the RRs were 1.13 (95% CI: 1.02, 1.24; P = 0.017) for breastfeeding in the first hour, 1.20 (95% CI: 1.06, 1.35; P = 0.004) for breast milk first fed after leaving hospital, and 1.49 (95% CI: 1.21, 1.84; P < 0.001) for 3-mo breastfeeding only, suggesting that prenatal perception was independently associated with breastfeeding outcomes. CONCLUSIONS: Prenatal perception of WIC's breastfeeding recommendations can be regarded as a new psychosocial predictor of breastfeeding and a possible target for future intervention.

Novel variants of engineered water soluble mu opioid receptors with extensive mutations and removal of cysteines.

We have shown that water-soluble variants of the human mu opioid receptor (wsMOR) containing a reduced number of hydrophobic residues at the lipid-facing residues of the transmembrane (TM) helices can be expressed in E. coli. In this study, we tested the consequences of increasing the number of mutations on the surface of the transmembrane domain on the receptor's aqueous solubility and ligand binding properties, along with mutation of 11 cysteine residues regardless of their solvent exposure value and location in the protein. We computationally engineered 10 different variants of MOR, and tested four of them for expression in E. coli. We found that all four variants were successfully expressed and could be purified in high quantities. The variants have alpha helical structural content similar to that of the native MOR, and they also display binding affinities for the MOR antagonist (naltrexone) similar to the wsMOR variants we engineered previously that contained many fewer mutations. Furthermore, for these full-length variants, the helical content remains unchanged over a wide range of pH values (pH 6 ~ 9). This study demonstrates the flexibility and robustness of the water-soluble MOR variants with respect to additional designed mutations in the TM domain and changes in pH, whereupon the protein's structural integrity and its ligand binding affinity are maintained. These variants of the full-length MOR with less hydrophobic surface residues and less cysteines can be obtained in large amounts from expression in E. coli and can serve as novel tools to investigate structure-function relationships of the receptor.

Variations in utilization and clinical outcomes for endoscopic sphenopalatine artery ligation and endovascular arterial embolization in a single multi-hospital network.

PURPOSE: Endoscopic sphenopalatine artery ligation (ESPAL) and endovascular arterial embolization (EAE) are increasingly common treatment options for patients with refractory epistaxis. The objective of this study was to compare the utilization pattern and clinical outcomes between these interventions within our single multi-hospital network. MATERIALS AND METHODS: A retrospective study of all patients undergoing ESPAL and/or EAE within any of the hospitals in a single healthcare network between 2008 and 2017 was conducted. We compared differences in procedure utilization with various hospital characteristics. Secondarily, we evaluated clinical outcomes and costs associated with each procedure. RESULTS: Forty-three ESPAL and 33 EAE procedures were performed across 7 hospitals, with the majority of procedures being performed at teaching institutions (65% and 91%, p = .013). The majority of both interventions were performed in larger hospitals and EAE patients were more likely to undergo inter-hospital transfer compared to ESPAL patients (48.5% and 16.3%, p = .02). Success rates for ESPAL and EAE were comparable (95% and 93%); however, the median direct cost of treatment for EAE was significantly higher than the cost for ESPAL ($12984.89 and $5002.02, p < .0001). CONCLUSIONS: The majority of both ESPAL and EAE interventions were performed at teaching and larger hospitals. Transfers occurring prior to EAE may have been due to the limited availability of interventional radiology services, and likely contributed to the increased cost of treatment. ESPAL is a known cost-effective management strategy and should be considered early in treatment algorithms of refractory epistaxis.

Combination of resveratrol and green tea epigallocatechin gallate induces synergistic apoptosis and inhibits tumor growth in vivo in head and neck cancer models.

Despite widespread interest in chemoprevention and therapy due to the high margin of safety of dietary natural compounds, clinical intervention with single agents has failed to yield the expected outcomes, mostly due to poor bioavailability and low potency. Combinations of natural agents with synergistic effects are gaining increasing attention. In the present study, in vitro and in vivo antitumor effects of a combination of two natural dietary agents, green tea epigallocatechin gallate (EGCG) and resveratrol were investigated. It was revealed that their combination at low doses (at which single agents induce minimal apoptosis) synergistically increased apoptosis (combination index < 1) in head and neck cancer cell lines. Synergistic apoptosis was also supported by caspase­3 and PARP cleavage. The combination also significantly inhibited growth of xenografted head and neck tumors in nude mice as supported by significant inhibition of tumor volume, tumor weight and Ki67 expression, and increase in TUNEL­positive cells. Mechanistic studies revealed that the combination inhibited AKT­mTOR signaling both in vitro and in vivo. In addition, overexpression of constitutively active AKT protected cells from apoptosis induced by the combination of EGCG and resveratrol. Collectively, the present results for the first time suggest that the combination of EGCG and resveratrol has synergistic growth inhibitory effects and provide an important rationale for future clinical development for chemoprevention and treatment of head and neck cancer.

Elucidating Protein Translocon Dynamics with Single-Molecule Precision.

Translocons are protein assemblies that facilitate the targeting and transport of proteins into and across biological membranes. Our understanding of these systems has been advanced using genetics, biochemistry, and structural biology. Despite these classic advances, until recently we have still largely lacked a detailed understanding of how translocons recognize and facilitate protein translocation. With the advent and improvements of cryogenic electron microscopy (cryo-EM) single-particle analysis and single-molecule fluorescence microscopy, the details of how translocons function are finally emerging. Here, we introduce these methods and evaluate their importance in understanding translocon structure, function, and dynamics.

Single-molecule view of coordination in a multi-functional DNA polymerase.

Replication and repair of genomic DNA requires the actions of multiple enzymatic functions that must be coordinated in order to ensure efficient and accurate product formation. Here, we have used single-molecule FRET microscopy to investigate the physical basis of functional coordination in DNA polymerase I (Pol I) from Escherichia coli, a key enzyme involved in lagging-strand replication and base excision repair. Pol I contains active sites for template-directed DNA polymerization and 5' flap processing in separate domains. We show that a DNA substrate can spontaneously transfer between polymerase and 5' nuclease domains during a single encounter with Pol I. Additionally, we show that the flexibly tethered 5' nuclease domain adopts different positions within Pol I-DNA complexes, depending on the nature of the DNA substrate. Our results reveal the structural dynamics that underlie functional coordination in Pol I and are likely relevant to other multi-functional DNA polymerases.

Discrimination between Functional and Non-functional Cellular Gag Complexes involved in HIV-1 Assembly.

HIV-1 Gag and Gag-Pol are responsible for viral assembly and maturation and represent a major paradigm for enveloped virus assembly. Numerous intracellular Gag-containing complexes (GCCs) have been identified in cellular lysates using sucrose gradient ultracentrifugation. While these complexes are universally present in Gag-expressing cells, their roles in virus assembly are not well understood. Here we demonstrate that most GCC species are predominantly comprised of monomeric or dimeric Gag molecules bound to ribosomal complexes, and as such, are not on-pathway intermediates in HIV assembly. Rather, these GCCs represent a population of Gag that is not yet functionally committed for incorporation into a viable virion precursor. We hypothesize that these complexes act as a reservoir of monomeric Gag that can incorporate into assembling viruses, and serve to mitigate non-specific intracellular Gag oligomerization. We have identified a subset of large GCC complexes, comprising more than 20 Gag molecules, that may be equivalent to membrane-associated puncta previously shown to be bona fide assembling-virus intermediates. This work provides a clear rationale for the existence of diverse GCCs, and serves as the foundation for characterizing on-pathway intermediates early in virus assembly.