Design, Synthesis, and Evaluation of a New Chemotype Fluorescent Ligand for the P2Y2 Receptor.

The P2Y2 receptor (P2Y2R) is a target for diseases including cancer, idiopathic pulmonary fibrosis, and atherosclerosis. However, there are insufficient P2Y2R antagonists available for validating P2Y2R function and future drug development. Evaluation of how (R)-5-(7-chloro-2-((2-ethoxyethyl)amino)-4H-benzo[5,6]cyclohepta[1,2-d]thiazol-4-yl)-1-methyl-4-thioxo-3,4-dihydropyrimidin-2(1H)-one, a previously published thiazole-based analogue of AR-C118925, binds in a P2Y2R homology model was used to design new P2Y2R antagonist scaffolds. One P2Y2R antagonist scaffold retained millimolar affinity for the P2Y2R and upon further functionalization with terminal carboxylic acid groups affinity was improved over 100-fold. This functionalized P2Y2R antagonist scaffold was employed to develop new chemotype P2Y2R fluorescent ligands, that were attainable in a convergent five-step synthesis. One of these fluorescent ligands demonstrated micromolar affinity (pK d = 6.02 ± 0.12, n = 5) for the P2Y2R in isolated cell membranes and distinct pharmacology from an existing P2Y2R fluorescent antagonist, suggesting it may occupy a different binding site on the P2Y2R.

Ligand-Directed Labeling of the Adenosine A1 Receptor in Living Cells.

The study of protein function and dynamics in their native cellular environment is essential for progressing fundamental science. To overcome the requirement of genetic modification of the protein or the limitations of dissociable fluorescent ligands, ligand-directed (LD) chemistry has most recently emerged as a complementary, bioorthogonal approach for labeling native proteins. Here, we describe the rational design, development, and application of the first ligand-directed chemistry approach for labeling the A1AR in living cells. We pharmacologically demonstrate covalent labeling of A1AR expressed in living cells while the orthosteric binding site remains available. The probes were imaged using confocal microscopy and fluorescence correlation spectroscopy to study A1AR localization and dynamics in living cells. Additionally, the probes allowed visualization of the specific localization of A1ARs endogenously expressed in dorsal root ganglion (DRG) neurons. LD probes developed here hold promise for illuminating ligand-binding, receptor signaling, and trafficking of the A1AR in more physiologically relevant environments.

A novel and selective fluorescent ligand for the study of adenosine A2B receptors.

Fluorescent ligands have proved to be powerful tools in the study of G protein-coupled receptors in living cells. Here we have characterized a new fluorescent ligand PSB603-BY630 that has high selectivity for the human adenosine A2B receptor (A2BR). The A2BR appears to play an important role in regulating immune responses in the tumor microenvironment. Here we have used PSB603-BY630 to monitor specific binding to A2BRs in M1- and M2-like macrophages derived from CD14+ human monocytes. PSB603-BY630 bound with high affinity (18.3 nM) to nanoluciferase-tagged A2BRs stably expressed in HEK293G cells. The ligand exhibited very high selectivity for the A2BR with negligible specific-binding detected at NLuc-A2AR, NLuc-A1R, or NLuc-A3R receptors at concentrations up to 500 nM. Competition binding studies showed the expected pharmacology at A2BR with the A2BR-selective ligands PSB603 and MRS-1706 demonstrating potent inhibition of the specific binding of 50 nM PSB603-BY630 to A2BR. Functional studies in HEK293G cells using Glosensor to monitor Gs-coupled cyclic AMP responses indicated that PSB603-BY630 acted as a negative allosteric regular of the agonist responses to BAY 60-6583. Furthermore, flow cytometry analysis confirmed that PSB603-BY630 could be used to selectively label endogenous A2BRs expressed on human macrophages. This ligand should be an important addition to the library of fluorescent ligands which are selective for the different adenosine receptor subtypes, and will enable study of the role of A2BRs on immune cells in the tumor microenvironment.

Spin-Phonon Coupling in Iron-Doped Ultrathin Bismuth Halide Perovskite Derivatives.

Spin in semiconductors facilitates magnetically controlled optoelectronic and spintronic devices. In metal halide perovskites (MHPs), doping magnetic ions is proven to be a simple and efficient approach to introducing a spin magnetic momentum. In this work, we present a facile metal ion doping protocol through the vapor-phase metal halide insertion reaction to the chemical vapor deposition (CVD)-grown ultrathin Cs3BiBr6 perovskites. The Fe-doped bismuth halide (Fe:CBBr) perovskites demonstrate that the iron spins are successfully incorporated into the lattice, as revealed by the spin-phonon coupling below the critical temperature Tc around 50 K observed through temperature-dependent Raman spectroscopy. Furthermore, the phonons exhibit significant softening under an applied magnetic field, possibly originating from magnetostriction and spin exchange interaction. The spin-phonon coupling in Fe:CBBr potentially provides an efficient way to tune the spin and lattice parameters for halide perovskite-based spintronics.

Mechano-sensitivity of ß2-adrenoceptors enhances constitutive activation of cAMP generation that is inhibited by inverse agonists.

The concept of agonist-independent signalling that can be attenuated by inverse agonists is a fundamental element of the cubic ternary complex model of G protein-coupled receptor (GPCR) activation. This model shows how a GPCR can exist in two conformational states in the absence of ligands; an inactive R state and an active R* state that differ in their affinities for agonists, inverse agonists, and G-protein alpha subunits. The proportion of R* receptors that exist in the absence of agonists determines the level of constitutive receptor activity. In this study we demonstrate that mechanical stimulation can induce ß2-adrenoceptor agonist-independent Gs-mediated cAMP signalling that is sensitive to inhibition by inverse agonists such as ICI-118551 and propranolol. The size of the mechano-sensitive response is dependent on the cell surface receptor expression level in HEK293G cells, is still observed in a ligand-binding deficient D113A mutant ß2-adrenoceptor and can be attenuated by site-directed mutagenesis of the extracellular N-glycosylation sites on the N-terminus and second extracellular loop of the ß2-adrenoceptor. Similar mechano-sensitive agonist-independent responses are observed in HEK293G cells overexpressing the A2A-adenosine receptor. These data provide new insights into how agonist-independent constitutive receptor activity can be enhanced by mechanical stimulation and regulated by inverse agonists.

CXCL17 is an allosteric inhibitor of CXCR4 through a mechanism of action involving glycosaminoglycans.

CXCL17 is a chemokine principally expressed by mucosal tissues, where it facilitates chemotaxis of monocytes, dendritic cells, and macrophages and has antimicrobial properties. CXCL17 is also implicated in the pathology of inflammatory disorders and progression of several cancers, and its expression is increased during viral infections of the lung. However, the exact role of CXCL17 in health and disease requires further investigation, and there is a need for confirmed molecular targets mediating CXCL17 functional responses. Using a range of bioluminescence resonance energy transfer (BRET)-based assays, here we demonstrated that CXCL17 inhibited CXCR4-mediated signaling and ligand binding. Moreover, CXCL17 interacted with neuropillin-1, a VEGFR2 coreceptor. In addition, we found that CXCL17 only inhibited CXCR4 ligand binding in intact cells and demonstrated that this effect was mimicked by known glycosaminoglycan binders, surfen and protamine sulfate. Disruption of putative GAG binding domains in CXCL17 prevented CXCR4 binding. This indicated that CXCL17 inhibited CXCR4 by a mechanism of action that potentially required the presence of a glycosaminoglycan-containing accessory protein. Together, our results revealed that CXCL17 is an endogenous inhibitor of CXCR4 and represents the next step in our understanding of the function of CXCL17 and regulation of CXCR4 signaling.

Small Molecule Fluorescent Ligands for the Atypical Chemokine Receptor 3 (ACKR3).

The atypical chemokine receptor 3 (ACKR3) is a receptor that induces cancer progression and metastasis in multiple cell types. Therefore, new chemical tools are required to study the role of ACKR3 in cancer and other diseases. In this study, fluorescent probes, based on a series of small molecule ACKR3 agonists, were synthesized. Three fluorescent probes, which showed specific binding to ACKR3 through a luminescence-based NanoBRET binding assay (pKd ranging from 6.8 to 7.8) are disclosed. Due to their high affinity at the ACKR3, we have shown their application in both competition binding experiments and confocal microscopy studies showing the cellular distribution of this receptor.

Role of endothelin ETA receptors in the hypertension induced by the VEGFR-2 kinase inhibitors axitinib and lenvatinib in conscious freely-moving rats.

Receptor tyrosine kinase inhibitors (RTKIs) suppress tumour growth by targeting vascular endothelial growth factor receptor 2 (VEGFR-2) which is an important mediator of angiogenesis. Here, we demonstrate that two potent RTKIs, axitinib and lenvatinib, are associated with hypertensive side effects. Doppler flowmetry was used to evaluate regional haemodynamic profiles of axitinib and lenvatinib. Male Sprague Dawley rats (350-500 g) were instrumented with Doppler flow probes (renal and mesenteric arteries and descending abdominal aorta) and catheters (jugular vein and distal abdominal aorta, via the caudal artery). Rats were dosed daily with axitinib (3 or 6 mg.kg-1) or lenvatinib (1 or 3 mg.kg-1) and regional haemodynamics were recorded over a maximum of 4 days. Both RTKIs caused significant (p < 0.05) increases in mean arterial pressure (MAP), which was accompanied by significant (p < 0.05) vasoconstriction in both the mesenteric and hindquarters vascular beds. To gain insight into the involvement of endothelin-1 (ET-1) in RTKI-mediated hypertension, we also monitored heart rate (HR) and MAP in response to axitinib or lenvatinib in animals treated with the ETA receptor selective antagonist sitaxentan (5 mg.kg-1) or the mixed ETA/ETB receptor antagonist bosentan (15 mg.kg-1) over two days. Co-treatment with bosentan or sitaxentan markedly reduced the MAP effects mediated by both RTKIs (p < 0.05). Bosentan, but not sitaxentan, also attenuated ET-1 mediated increases in HR. These data suggest that selective antagonists of ETA receptors may be appropriate to alleviate the hypertensive effects of axitinib and lenvatinib.

Characterisation of tyrosine kinase inhibitor-receptor interactions at VEGFR2 using sunitinib-red and nanoBRET.

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis, proliferation and migration of vascular endothelial cells. It is well known that cardiovascular safety liability for a wide range of small molecule tyrosine kinase inhibitors (TKIs) can result from interference with the VEGFR2 signalling system. In this study we have developed a ligand-binding assay using a fluorescent analogue of sunitinib (sunitinib-red) and full length VEGFR2 tagged on its C-terminus with the bioluminescent protein nanoluciferase to monitor ligand-binding to VEGFR2 using bioluminescence resonance energy transfer (BRET). This NanoBRET assay is a proximity-based assay (requiring the fluorescent and bioluminescent components to be within 10 nm of each other) that can monitor the binding of ligands to the kinase domain of VEGFR2. Sunitinib-red was not membrane permeable but was able to monitor the binding affinity and kinetics of a range of TKIs in cell lysates. Kinetic studies showed that sunitinib-red bound rapidly to VEGFR2 at 25 °C and that cediranib had slower binding kinetics with an average residence time of 111 min. Comparison between the log Ki values for inhibition of binding of sunitinib-red and log IC50 values for attenuation of VEGF165a-stimulated NFAT responses showed very similar values for compounds that inhibited sunitinib-red binding. However, two compounds that failed to inhibit sunitinib-red binding (dasatinib and entospletinib) were still able to attenuate VEGFR2-mediated NFAT signalling through inhibition of downstream signalling events. These results suggest that these compounds may still exhibit cardiovascular liabilities as a result of interference with downstream VEGFR2 signalling.

The evolution of dialysis access surgery over 38 years: One surgeon's perspective.

Objectives: The surgery for dialysis access has changed over the past 38 years. In the 1980s and 1990s, prosthetic grafts were the most common form of access. Then, autogenous fistulae had a rebirth due to their durability and decreased complications. The continued expansion of the dialysis population, coupled with the paucity of adequate superficial veins in many patients, required other techniques of dialysis access such as tunneled dialysis catheters, and more complex surgery on deeper veins. Methods: This study of one surgeon's practice over 38 years mirrors the extensive changes in dialysis access. The changes in surgical technique, interventional procedures, and approaches were documented and evaluated. Results: During the 38-year period, there were 1531 autogenous fistulae, 409 prosthetic grafts, and 1624 tunneled dialysis catheters placed for access. The first 20 years had 130 autogenous fistulae with 302 prosthetic grafts, while in the last 10 years there were 740 fistulae and only 17 prosthetic grafts. Prosthetic grafts were not salvageable for a long term with exposure, infection, and persistent bleeding. Autogenous fistulae were best salvaged with autogenous tissue rather than prosthetic material. Interventional procedures were most valuable in stenting high-grade stenosis centrally and dilating areas of recurrent stenosis. They were not helpful in treatment of large aneurysms or as a long-term solution for persistent and/or massive bleeding. Conclusion: Dialysis access has progressed back to autogenous fistula. This may require longer use of tunneled dialysis catheters, and more surgical procedures, but the construction of an autogenous fistula can be achieved in many dialysis patients.

Small-Molecule Fluorescent Ligands for the CXCR4 Chemokine Receptor.

The C-X-C chemokine receptor type 4, or CXCR4, is a chemokine receptor found to promote cancer progression and metastasis of various cancer cell types. To investigate the pharmacology of this receptor, and to further elucidate its role in cancer, novel chemical tools are a necessity. In the present study, using classic medicinal chemistry approaches, small-molecule-based fluorescent probes were designed and synthesized based on previously reported small-molecule antagonists. Here, we report the development of three distinct chemical classes of fluorescent probes that show specific binding to the CXCR4 receptor in a novel fluorescence-based NanoBRET binding assay (pKD ranging 6.6-7.1). Due to their retained affinity at CXCR4, we furthermore report their use in competition binding experiments and confocal microscopy to investigate the pharmacology and cellular distribution of this receptor.

Rapid chloride and bicarbonate determination by capillary electrophoresis for confirmatory testing of cystic fibrosis infants with volume-limited sweat specimens.

Objectives Cystic fibrosis (CF) is a debilitating genetic disorder that benefits from early detection. CF diagnosis relies on measuring elevated sweat chloride that is difficult in neonates with low sweat rates. We introduce a new method for sweat chloride determination from volume-limited specimens, and explore the potential utility of sweat bicarbonate in neonatal CF screening. Methods A rapid assay (< 5 min) was developed to analyze chloride and bicarbonate using capillary electrophoresis with indirect UV detection (CE-iUV). Pilocarpine-stimulated sweat samples from screen-positive CF infants were collected at two hospital sites, including confirmed CF (n = 12), CF screen-positive inconclusive diagnosis (n = 4), and unaffected non-CF cases (n = 37). All sweat chloride samples were analyzed by a coulometric titrator and CE-iUV, and the viability to measure acid-labile bicarbonate was also evaluated. Results Stability studies revealed that bicarbonate can be reliably assessed in sweat if acidification and heating were avoided. Method validation demonstrated that sweat chloride and bicarbonate were quantified with acceptable accuracy (recovery of 102%), precision (CV = 3.7%) and detection limits (∼ 0.1 mM). An inter-laboratory comparison confirmed a mean bias of 6.5% (n = 53) for sweat chloride determination by CE-iUV relative to a commercial chloridometer. However, sweat bicarbonate did not discriminate between CF and non-CF infants (AUC = 0.623, p = 0.215) unlike chloride (AUC = 1.00, p = 3.00 × 10-7). Conclusions CE-iUV offers a robust method for sweat chloride testing from presumptive CF infants that may reduce testing failure rates. However, sweat bicarbonate does not have clinical value in newborn CF diagnosis.

Kinetic analysis of endogenous ß2 -adrenoceptor-mediated cAMP GloSensor™ responses in HEK293 cells.

BACKGROUND AND AIM: Standard pharmacological analysis of agonist activity utilises measurements of receptor-mediated responses at a set time-point, or at the peak response level, to characterise ligands. However, the occurrence of non-equilibrium conditions may dramatically impact the properties of the response being measured. Here we have analysed the initial kinetic phases of cAMP responses to ß2 -adrenoceptor agonists in HEK293 cells expressing the endogenous ß2 -adrenoceptor at extremely low levels. EXPERIMENTAL APPROACH: The kinetics of ß2 -adrenoceptor agonist-stimulated cAMP responses were monitored in real-time, in the presence and absence of antagonists, in HEK293 cells expressing the cAMP GloSensor™ biosensor. Potency (EC50 ) and efficacy (Emax ) values were determined at the peak of the agonist GloSensor™ response and compared to kinetic parameters L50 and IRmax values derived from initial response rates. KEY RESULTS: The partial agonists salbutamol and salmeterol displayed reduced relative IRmax values (with respect to isoprenaline) when compared with their Emax values. Except for the fast dissociating bisoprolol, preincubation with ß2 -adrenoceptor antagonists produced a large reduction in the isoprenaline peak response due to a state of hemi-equilibrium in this low receptor reserve system. This effect was exacerbated when IRmax parameters were measured. Furthermore, bisoprolol produced a large reduction in isoprenaline IRmax consistent with its short residence time. CONCLUSIONS AND IMPLICATIONS: Kinetic analysis of real-time signalling data can provide valuable insights into the hemi-equilibria that can occur in low receptor reserve systems with agonist-antagonist interactions, due to incomplete dissociation of antagonist whilst the peak agonist response is developing.

Fluorescently tagged nanobodies and NanoBRET to study ligand-binding and agonist-induced conformational changes of full-length EGFR expressed in living cells.

Introduction: The Epidermal Growth Factor Receptor is a member of the Erb receptor tyrosine kinase family. It binds several ligands including EGF, betacellulin (BTC) and TGF-α, controls cellular proliferation and invasion and is overexpressed in various cancer types. Nanobodies (VHHs) are the antigen binding fragments of heavy chain only camelid antibodies. In this paper we used NanoBRET to compare the binding characteristics of fluorescent EGF or two distinct fluorescently labelled EGFR directed nanobodies (Q44c and Q86c) to full length EGFR. Methods: Living HEK293T cells were stably transfected with N terminal NLuc tagged EGFR. NanoBRET saturation, displacement or kinetics experiments were then performed using fluorescently labelled EGF ligands (EGF-AF488 or EGF-AF647) or fluorescently labelled EGFR targeting nanobodies (Q44c-HL488 and Q86c-HL488). Results: These data revealed that the EGFR nanobody Q44c was able to inhibit EGF binding to full length EGFR, while Q86c was able to recognise agonist bound EGFR and act as a conformational sensor. The specific binding of fluorescent Q44c-HL488 and EGF-AF488 was inhibited by a range of EGFR ligands (EGF> BTC>TGF-α). Discussion: EGFR targeting nanobodies are powerful tools for studying the role of the EGFR in health and disease and allow real time quantification of ligand binding and distinct ligand induced conformational changes.

Involvement of ß-adrenoceptors in the cardiovascular responses induced by selective adenosine A2A and A2B receptor agonists.

A2A and A2B adenosine receptors produce regionally selective regulation of vascular tone and elicit differing effects on mean arterial pressure (MAP), whilst inducing tachycardia. The tachycardia induced by the stimulation of A2A or A2B receptors has been suggested to be mediated by a reflex increase in sympathetic activity. Here, we have investigated the role of ß1 - and ß2 -adrenoceptors in mediating the different cardiovascular responses to selective A2A and A2B receptor stimulation. Hemodynamic variables were measured in conscious male Sprague-Dawley rats (350-450 g) via pulsed Doppler flowmetry. The effect of intravenous infusion (3 min per dose) of the A2A -selective agonist CGS 21680 (0.1, 0.3, 1.0 µg.kg-1 .min-1 ) or the A2B -selective agonist BAY 60-6583 (4.0, 13.3, 40.0 µg.kg-1 .min-1 ) in the absence or following pre-treatment with the non-selective ß-antagonist propranolol (1.0 mg.kg-1 ), the selective ß1 -antagonist CGP 20712A (200 µg.kg-1 ), or the selective ß2 -antagonist ICI 118,551 (2.0 mg.kg-1 ) was investigated (maintenance doses also administered). CGP 20712A and propranolol significantly reduced the tachycardic response to CGS 21680, with no change in the effect on MAP. ICI 118,551 increased BAY 60-6583-mediated renal and mesenteric flows, but did not affect the heart rate response. CGP 20712A attenuated the BAY 60-6583-induced tachycardia. These data imply a direct stimulation of the sympathetic activity via cardiac ß1 -adrenoceptors as a mechanism for the A2A - and A2B -induced tachycardia. However, the regionally selective effects of A2B agonists on vascular conductance were independent of sympathetic activity and may be exploitable for the treatment of acute kidney injury and mesenteric ischemia.

Optimization of Peptide Linker-Based Fluorescent Ligands for the Histamine H1 Receptor.

The histamine H1 receptor (H1R) has recently been implicated in mediating cell proliferation and cancer progression; therefore, high-affinity H1R-selective fluorescent ligands are desirable tools for further investigation of this behavior in vitro and in vivo. We previously reported a H1R fluorescent ligand, bearing a peptide-linker, based on antagonist VUF13816 and sought to further explore structure-activity relationships (SARs) around the linker, orthostere, and fluorescent moieties. Here, we report a series of high-affinity H1R fluorescent ligands varying in peptide linker composition, orthosteric targeting moiety, and fluorophore. Incorporation of a boron-dipyrromethene (BODIPY) 630/650-based fluorophore conferred high binding affinity to our H1R fluorescent ligands, remarkably overriding the linker SAR observed in corresponding unlabeled congeners. Compound 31a, both potent and subtype-selective, enabled H1R visualization using confocal microscopy at a concentration of 10 nM. Molecular docking of 31a with the human H1R predicts that the optimized peptide linker makes interactions with key residues in the receptor.

Regionally selective cardiovascular responses to adenosine A2A and A2B receptor activation.

Adenosine is a local mediator that regulates changes in the cardiovascular system via activation of four G protein-coupled receptors (A1 , A2A , A2B , A3 ). Here, we have investigated the effect of A2A and A2B -selective agonists on vasodilatation in three distinct vascular beds of the rat cardiovascular system. NanoBRET ligand binding studies were used to confirm receptor selectivity. The regional hemodynamic effects of adenosine A2A and A2B selective agonists were investigated in conscious rats. Male Sprague-Dawley rats (350-450 g) were chronically implanted with pulsed Doppler flow probes on the renal artery, mesenteric artery, and the descending abdominal aorta. Cardiovascular responses were measured following intravenous infusion (3 min for each dose) of the A2A -selective agonist CGS 21680 (0.1, 0.3, 1 µg kg-1 min-1 ) or the A2B -selective agonist BAY 60-6583 (4,13.3, 40 µg kg-1 min-1 ) following predosing with the A2A -selective antagonist SCH 58261 (0.1 or 1 mg kg-1 min-1 ), the A2B /A2A antagonist PSB 1115 (10 mg kg-1 min-1 ) or vehicle. The A2A -selective agonist CGS 21680 produced a striking increase in heart rate (HR) and hindquarters vascular conductance (VC) that was accompanied by a significant decrease in mean arterial pressure (MAP) in conscious rats. In marked contrast, the A2B -selective agonist BAY 60-6583 significantly increased HR and VC in the renal and mesenteric vascular beds, but not in the hindquarters. Taken together, these data indicate that A2A and A2B receptors are regionally selective in their regulation of vascular tone. These results suggest that the development of A2B receptor agonists to induce vasodilatation in the kidney may provide a good therapeutic approach for the treatment of acute kidney injury.

Ureteral stenting after routine ureteroscopy: Is earlier stent removal feasible?

INTRODUCTION: Ureteroscopy and laser lithotripsy is a common treatment option for upper urinary tract calculi. Currently, ureteral stents are placed after uncomplicated ureteroscopy for up to 1 week, but the optimal length of placement is not well defined. Ureteral stents are associated with significant morbidity, particularly stent discomfort. This study aims to determine differences in postoperative unplanned clinic or ED visits based on duration of stent placement. MATERIALS AND METHODS: This is a single-institution, IRB-approved, retrospective cohort study of 559 ureteroscopy cases with laser lithotripsy for urinary tract calculi performed from 2016 to 2018. The primary outcome was unplanned ED or clinic visits within 30 days following surgery and there. The patients were separated into three groups based on stent duration: 1 (0-3 days), 2 (4-6 days), and 3 (> 6 days). RESULTS: Fifty-eight (10.31%) patients experienced an unplanned visit within 30 days of the procedure. There was no significant difference in unplanned visits among groups for stent duration (p = 0.45). A Clavien grade analysis showed no difference in grades between groups (p = 0.59). A Cox regression model showed no difference in risk of unplanned visit comparing those in groups 2 and 3 to group 1 (p = 0.157 and 0.374, respectively). This also remains to be the case after adjusting for age, sex, and surgeon (p = 0.166 and 0.376, respectively). CONCLUSIONS: We found no difference in unplanned visits in patients based on the duration of stent placement following routine ureteroscopy. Stent removal within 3 days of surgery appears to be sufficient to minimize morbidity after uncomplicated ureteroscopy.

Isolation of a triplet benzene dianion.

Baird's rule predicts that molecules with 4n π electrons should be aromatic in the triplet state, but the realization of simple ring systems with such an electronic ground state has been stymied by these molecules' tendency to distort into structures bearing a large singlet-triplet gap. Here, we show that the elusive benzene diradical dianion can be stabilized through creation of a binucleating ligand that enforces a tightly constrained inverse sandwich structure and direct magnetic exchange coupling. Specifically, we report the compounds [K(18-crown-6)(THF)2]2[M2(BzN6-Mes)] (M = Y, Gd; BzN6-Mes = 1,3,5-tris[2',6'-(N-mesityl)dimethanamino-4'-tert-butylphenyl]benzene), which feature a trigonal ligand that binds one trivalent metal ion on each face of a central benzene dianion. Antiferromagnetic exchange in the Gd3+ compound preferentially stabilizes the triplet state such that it becomes the molecular ground state. Single-crystal X-ray diffraction data and nucleus-independent chemical shift calculations support aromaticity, in agreement with Baird's rule.

Development of Covalent, Clickable Probes for Adenosine A1 and A3 Receptors.

Adenosine receptors are attractive therapeutic targets for multiple conditions, including ischemia-reperfusion injury and neuropathic pain. Adenosine receptor drug discovery efforts would be facilitated by the development of appropriate tools to assist in target validation and direct receptor visualization in different native environments. We report the development of the first bifunctional (chemoreactive and clickable) ligands for the adenosine A1 receptor (A1R) and adenosine A3 receptor (A3R) based on an orthosteric antagonist xanthine-based scaffold and on an existing structure-activity relationship. Bifunctional ligands were functional antagonists with nanomolar affinity and irreversible binding at the A1R and A3R. In-depth pharmacological profiling of these bifunctional ligands showed moderate selectivity over A2A and A2B adenosine receptors. Once bound to the receptor, ligands were successfully "clicked" with a cyanine-5 fluorophore containing the complementary "click" partner, enabling receptor detection. These bifunctional ligands are expected to aid in the understanding of A1R and A3R localization and trafficking in native cells and living systems.