Sidestream bio-P and mainstream anammox in a BNR process with upstream carbon capture.

The integration of biological phosphorus removal (bio-P) and shortcut nitrogen removal (SNR) processes is challenging because of the conflicting demands on influent carbon: SNR allows for upstream carbon diversion, but this reduction of influent carbon (especially volatile fatty acids [VFAs]) prevents or limits bio-P. The objective of this study was to achieve SNR, either via partial nitritation/anammox (PNA) or partial denitrification/anammox (PdNA), simultaneously with biological phosphorus removal in a process with upstream carbon capture. This study took place in a pilot scale A/B process with a sidestream bio-P reactor and tertiary anammox polishing. Despite low influent rbCOD concentrations from the A-stage effluent, bio-P occurred in the B-stage thanks to the addition of A-stage WAS fermentate to the sidestream reactor. Nitrite accumulation occurred in the B-stage via partial denitrification and partial nitritation (NOB out-selection), depending on operational conditions, and was removed along with ammonia by the tertiary anammox MBBR, with the ability to achieve effluent TIN less than 2 mg/L. PRACTITIONER POINTS: A sidestream reactor with sufficient fermentate addition enables biological phosphorus removal in a B-stage system with little-to-no influent VFA. Enhanced biological phosphorus removal is not inhibited by intermittent aeration and is stable at a wide range of process SRTs. Partial nitritation and partial denitrification are viable routes to produce nitrite within an A/B process with sidestream bio-P, for downstream anammox in a polishing MBBR.

Bees and thrips carry virus-positive pollen in peach orchards in South Carolina, United States.

Prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) are pollen-borne viruses of important stone fruit crops, including peaches, which can cause substantial yield loss. Although both horizontal and vertical (i.e., seed) transmission of both viruses occurs through pollen, the role of flower-visiting insects in their transmission is not well understood. Bees and thrips reportedly spread PNRSV and PDV in orchards and greenhouse studies; however, the field spread of PNRSV and PDV in peach orchards in the southeastern United States is not explored. We hypothesized that bees and thrips may facilitate virus spread by carrying virus-positive pollen. Our 2-yr survey results show that 75% of captured bees are carrying virus-positive pollen and moving across the orchard while a subsample of thrips were also found virus positive. Based on morphology, Bombus, Apis, Andrena, Eucera, and Habropoda are the predominant bee genera that were captured in peach orchards. Understanding the role of bees and thrips in the spread of PNRSV and PDV will enhance our understanding of pollen-borne virus ecology.

Enhancing upper-limb neurorehabilitation in chronic stroke survivors using combined action observation and motor imagery therapy.

Introduction: For people who have had a stroke, recovering upper-limb function is a barrier to independence. When movement is difficult, mental practice can be used to complement physical therapy. In this within-participants study we investigated the effects of combined action observation and motor imagery (AO + MI) therapy on upper-limb recovery in chronic stroke survivors. Methods: A Graeco-Latin Square design was used to counterbalance four mental practice conditions (AO + MI, AO, MI, Control) across four cup-stacking tasks of increasing complexity. Once a week, for five consecutive weeks, participants (n = 10) performed 16 mental practice trials under each condition. Each trial displayed a 1st person perspective of a cup-stacking task performed by an experienced model. For AO, participants watched each video and responded to an occasional color cue. For MI, participants imagined the effort and sensation of performing the action; cued by a series of still-images. For combined AO + MI, participants observed a video of the action while they simultaneously imagined performing the same action in real-time. At three time points (baseline; post-test; two-week retention test) participants physically executed the three mentally practiced cup-stacking tasks, plus a fourth unpractised sequence (Control), as quickly and accurately as possible. Results: Mean movement execution times were significantly reduced overall in the post-test and the retention test compared to baseline. At retention, movement execution times were significantly shorter for combined AO + MI compared to both MI and the Control. Individual participants reported clinically important changes in quality of life (Stroke Impact Scale) and positive qualitative experiences of AO + MI (social validation). Discussion: These results indicate that when physical practice is unsuitable, combined AO + MI therapy could offer an effective adjunct for neurorehabilitation in chronic stroke survivors.

D-Cystine di(m)ethyl ester reverses the deleterious effects of morphine on ventilation and arterial blood gas chemistry while promoting antinociception.

We have identified thiolesters that reverse the negative effects of opioids on breathing without compromising antinociception. Here we report the effects of D-cystine diethyl ester (D-cystine diEE) or D-cystine dimethyl ester (D-cystine diME) on morphine-induced changes in ventilation, arterial-blood gas chemistry, A-a gradient (index of gas-exchange in the lungs) and antinociception in freely moving rats. Injection of morphine (10 mg/kg, IV) elicited negative effects on breathing (e.g., depression of tidal volume, minute ventilation, peak inspiratory flow, and inspiratory drive). Subsequent injection of D-cystine diEE (500 µmol/kg, IV) elicited an immediate and sustained reversal of these effects of morphine. Injection of morphine (10 mg/kg, IV) also elicited pronounced decreases in arterial blood pH, pO2 and sO2 accompanied by pronounced increases in pCO2 (all indicative of a decrease in ventilatory drive) and A-a gradient (mismatch in ventilation-perfusion in the lungs). These effects of morphine were reversed in an immediate and sustained fashion by D-cystine diME (500 µmol/kg, IV). Finally, the duration of morphine (5 and 10 mg/kg, IV) antinociception was augmented by D-cystine diEE. D-cystine diEE and D-cystine diME may be clinically useful agents that can effectively reverse the negative effects of morphine on breathing and gas-exchange in the lungs while promoting antinociception. Our study suggests that the D-cystine thiolesters are able to differentially modulate the intracellular signaling cascades that mediate morphine-induced ventilatory depression as opposed to those that mediate morphine-induced antinociception and sedation.

Identification of Plasmodium dipeptidyl aminopeptidase allosteric inhibitors by high throughput screening.

Dipeptidyl aminopeptidases (DPAPs) are cysteine proteases that cleave dipeptides from the N-terminus of protein substrates and have been shown to play important roles in many pathologies including parasitic diseases such as malaria, toxoplasmosis and Chagas's disease. Inhibitors of the mammalian homologue cathepsin C have been used in clinical trials as potential drugs to treat chronic inflammatory disorders, thus proving that these enzymes are druggable. In Plasmodium species, DPAPs play important functions at different stages of parasite development, thus making them potential antimalarial targets. Most DPAP inhibitors developed to date are peptide-based or peptidomimetic competitive inhibitors. Here, we used a high throughput screening approach to identify novel inhibitor scaffolds that block the activity of Plasmodium falciparum DPAP1. Most of the hits identified in this screen also inhibit Plasmodium falciparum DPAP3, cathepsin C, and to a lesser extent other malarial clan CA proteases, indicating that these might be general DPAP inhibitors. Interestingly, our mechanism of inhibition studies indicate that most hits are allosteric inhibitors, which opens a completely new strategy to inhibit these enzymes, study their biological function, and potentially develop new inhibitors as starting points for drug development.

Clinical Outcomes After Physical Therapy Treatment for Secondary Lymphedema After Breast Cancer.

Breast cancer is the most commonly diagnosed cancer in women and approximately 33% of survivors will develop lymphedema. Untreated lymphedema may be limb threatening or cause substantial functional limitations. The purpose of this case report is to detail the physical therapy (PT) management and outcomes for a patient with right upper extremity and breast lymphedema. The goal of this case report is to provide rehabilitation clinicians with an example of effective treatment management and the underlying treatment rationale. A 64-year-old female with stage 2A breast cancer underwent neoadjuvant chemotherapy, a lumpectomy with 18 lymph nodes removed, and radiation therapy. She subsequently developed secondary lymphedema of the right breast and upper extremity. Physical therapy interventions included instruction on a complete decongestive therapy program, which consists of manual lymphatic drainage and compression bandaging and exercises to improve shoulder range of motion (ROM), posture, and strength. As a result of PT, her right shoulder ROM and anthropometric measurements improved and the patient achieved independence with self-lymphatic massage and compression bandaging techniques to maintain these gains. This case report is unique as it details the clinical decision making required during a complex course of cancer care that necessitated adjustments to the PT plan of care for sustainable outcomes.

A systematic review of research investigating the physiological and psychological effects of combining Ginkgo biloba and Panax ginseng into a single treatment in humans: Implications for research design and analysis.

BACKGROUND AND PURPOSE: The traditional herbal supplements Panax ginseng and Ginkgo biloba are self-medicated by members of the general public and prescribed by healthcare professionals in some EU countries for numerous health complaints. Clinical evidence is mixed and mechanisms of action are not fully understood. There is clinical interest into the synergistic effects of combining both herbs. METHODS: We systematically review the literature investigating the effects of combination treatments on physiological and psychological outcomes in humans. We identified all studies meeting inclusion criteria: (a) written in English; (b) peer-reviewed; (c) conducted in humans; (d) including either a proprietary Panax ginseng/Ginkgo biloba treatment or a study preparation containing both; (e) placebo-controlled; (f) utilizing standardized extracts. We critically discuss each trial; calculate standardized effect sizes where possible and provide recommendations for research design and analysis. RESULTS: Eight studies were identified and all investigated a proprietary combination treatment, Gincosan® . Studies are of high quality and robust; however, practice effects, choice of statistical model, and reliance upon null-hypothesis significance testing hinder generalized estimates of effect. The most consistent results are benefits to aspects of the circulatory/cardiovascular system in patient populations and "secondary memory" performance in patient and healthy populations. Two studies demonstrate synergy in healthy populations following a single dose; however, synergy in patient populations and following repeated dosing has not yet been directly tested. CONCLUSIONS: A Panax ginseng and Ginkgo biloba combination treatment can improve aspects of physiological and cognitive function in humans; however, evidence for synergy requires further investigation and future research should directly investigate synergy following repeated dosing.

Functional Assessment of 2,177 U.S. and International Drugs Identifies the Quinoline Nitroxoline as a Potent Amoebicidal Agent against the Pathogen Balamuthia mandrillaris.

Balamuthia mandrillaris is a pathogenic free-living amoeba that causes a rare but almost always fatal infection of the central nervous system called granulomatous amoebic encephalitis (GAE). Two distinct forms of B. mandrillaris-a proliferative trophozoite form and a nonproliferative cyst form, which is highly resistant to harsh physical and chemical conditions-have been isolated from environmental samples worldwide and are both observed in infected tissue. Patients suffering from GAE are typically treated with aggressive and prolonged multidrug regimens that often include the antimicrobial agents miltefosine and pentamidine isethionate. However, survival rates remain low, and studies evaluating the susceptibility of B. mandrillaris to these compounds and other potential therapeutics are limited. To address the need for more-effective treatments, we screened 2,177 clinically approved compounds for in vitro activity against B. mandrillaris The quinoline antibiotic nitroxoline (8-hydroxy-5-nitroquinoline), which has safely been used in humans to treat urinary tract infections, was identified as a lead compound. We show that nitroxoline inhibits both trophozoites and cysts at low micromolar concentrations, which are within a pharmacologically relevant range. We compared the in vitro efficacy of nitroxoline to that of drugs currently used in the standard of care for GAE and found that nitroxoline is the most potent and selective inhibitor of B. mandrillaris tested. Furthermore, we demonstrate that nitroxoline prevents B. mandrillaris-mediated destruction of host cells in cultured fibroblast and primary brain explant models also at pharmacologically relevant concentrations. Taken together, our findings indicate that nitroxoline is a promising candidate for repurposing as a novel treatment of B. mandrillaris infections.IMPORTANCEBalamuthia mandrillaris is responsible for hundreds of reported cases of amoebic encephalitis, the majority of which have been fatal. Despite being an exceptionally deadly pathogen, B. mandrillaris is understudied, leaving many open questions regarding epidemiology, diagnosis, and treatment. Due to the lack of effective drugs to fight B. mandrillaris infections, mortality rates remain high even for patients receiving intensive care. This report addresses the need for new treatment options through a drug repurposing screen to identify novel B. mandrillaris inhibitors. The most promising candidate identified was the quinoline antibiotic nitroxoline, which has a long history of safe use in humans. We show that nitroxoline kills B. mandrillaris at pharmacologically relevant concentrations and exhibits greater potency and selectivity than drugs commonly used in the current standard of care. The findings that we present demonstrate the potential of nitroxoline to be an important new tool in the treatment of life-threatening B. mandrillaris infections.

Obesity-related cellular stressors regulate gonadotropin releasing hormone gene expression via c-Fos/AP-1.

Obesity is a risk factor for infertility, but mechanisms underlying this risk are unclear. Fertility is regulated by hypothalamic gonadotropin-releasing hormone, encoded by the Gnrh1 gene. Because obesity promotes endoplasmic reticulum (ER) stress, we sought to determine how tunicamycin-induced ER stress affected Gnrh1 gene expression in the mouse hypothalamic cell line GT1-7. Tunicamycin repressed expression of Gnrh1 in a PKC- and JNK-dependent manner, while upregulating expression of a known Gnrh1 repressor, Fos. Obesity is associated with increased circulating free fatty acids, and exposure to palmitate promoted ER stress and inflammation. Fos expression increased with palmitate dose, but Gnrh1 expression was upregulated with low-dose palmitate and repressed with high-dose palmitate. Using a small molecule inhibitor, we determined that AP-1 was required for Gnrh1 repression by high-dose palmitate or tunicamycin-induced ER stress. These findings suggest that hypogonadism driven by decreased hypothalamic GnRH may be a component of obesity-related infertility.

A Calibrated Method of Massage Therapy Decreases Systolic Blood Pressure Concomitant With Changes in Heart Rate Variability in Male Rats.

OBJECTIVE: The purpose of this study was to develop a method for applying calibrated manual massage pressures by using commonly available, inexpensive sphygmomanometer parts and validate the use of this approach as a quantitative method of applying massage therapy to rodents. METHODS: Massage pressures were monitored by using a modified neonatal blood pressure (BP) cuff attached to an aneroid gauge. Lightly anesthetized rats were stroked on the ventral abdomen for 5 minutes at pressures of 20 mm Hg and 40 mm Hg. Blood pressure was monitored noninvasively for 20 minutes following massage therapy at 5-minute intervals. Interexaminer reliability was assessed by applying 20 mm Hg and 40 mm Hg pressures to a digital scale in the presence or absence of the pressure gauge. RESULTS: With the use of this method, we observed good interexaminer reliability, with intraclass coefficients of 0.989 versus 0.624 in blinded controls. In Long-Evans rats, systolic BP dropped by an average of 9.86% ± 0.27% following application of 40 mm Hg massage pressure. Similar effects were seen following 20 mm Hg pressure (6.52% ± 1.7%), although latency to effect was greater than at 40 mm Hg. Sprague-Dawley rats behaved similarly to Long-Evans rats. Low-frequency/high-frequency ratio, a widely-used index of autonomic tone in cardiovascular regulation, showed a significant increase within 5 minutes after 40 mm Hg massage pressure was applied. CONCLUSIONS: The calibrated massage method was shown to be a reproducible method for applying massage pressures in rodents and lowering BP.

Ceapins are a new class of unfolded protein response inhibitors, selectively targeting the ATF6α branch.

The membrane-bound transcription factor ATF6α plays a cytoprotective role in the unfolded protein response (UPR), required for cells to survive ER stress. Activation of ATF6α promotes cell survival in cancer models. We used cell-based screens to discover and develop Ceapins, a class of pyrazole amides, that block ATF6α signaling in response to ER stress. Ceapins sensitize cells to ER stress without impacting viability of unstressed cells. Ceapins are highly specific inhibitors of ATF6α signaling, not affecting signaling through the other branches of the UPR, or proteolytic processing of its close homolog ATF6ß or SREBP (a cholesterol-regulated transcription factor), both activated by the same proteases. Ceapins are first-in-class inhibitors that can be used to explore both the mechanism of activation of ATF6α and its role in pathological settings. The discovery of Ceapins now enables pharmacological modulation all three UPR branches either singly or in combination.

A gene-expression screen identifies a non-toxic sumoylation inhibitor that mimics SUMO-less human LRH-1 in liver.

SUMO-modification of nuclear proteins has profound effects on gene expression. However, non-toxic chemical tools that modulate sumoylation in cells are lacking. Here, to identify small molecule sumoylation inhibitors we developed a cell-based screen that focused on the well-sumoylated substrate, human Liver Receptor Homolog-1 (hLRH-1, NR5A2). Our primary gene-expression screen assayed two SUMO-sensitive transcripts, APOC3 and MUC1, that are upregulated by SUMO-less hLRH-1 or by siUBC9 knockdown, respectively. A polyphenol, tannic acid (TA) emerged as a potent sumoylation inhibitor in vitro (IC50 = 12.8 µM) and in cells. TA also increased hLRH-1 occupancy on SUMO-sensitive transcripts. Most significantly, when tested in humanized mouse primary hepatocytes, TA inhibits hLRH-1 sumoylation and induces SUMO-sensitive genes, thereby recapitulating the effects of expressing SUMO-less hLRH-1 in mouse liver. Our findings underscore the benefits of phenotypic screening for targeting post-translational modifications, and illustrate the potential utility of TA for probing the cellular consequences of sumoylation.

Characterization of the novel and specific PI3Kα inhibitor NVP-BYL719 and development of the patient stratification strategy for clinical trials.

Somatic PIK3CA mutations are frequently found in solid tumors, raising the hypothesis that selective inhibition of PI3Kα may have robust efficacy in PIK3CA-mutant cancers while sparing patients the side-effects associated with broader inhibition of the class I phosphoinositide 3-kinase (PI3K) family. Here, we report the biologic properties of the 2-aminothiazole derivative NVP-BYL719, a selective inhibitor of PI3Kα and its most common oncogenic mutant forms. The compound selectivity combined with excellent drug-like properties translates to dose- and time-dependent inhibition of PI3Kα signaling in vivo, resulting in robust therapeutic efficacy and tolerability in PIK3CA-dependent tumors. Novel targeted therapeutics such as NVP-BYL719, designed to modulate aberrant functions elicited by cancer-specific genetic alterations upon which the disease depends, require well-defined patient stratification strategies in order to maximize their therapeutic impact and benefit for the patients. Here, we also describe the application of the Cancer Cell Line Encyclopedia as a preclinical platform to refine the patient stratification strategy for NVP-BYL719 and found that PIK3CA mutation was the foremost positive predictor of sensitivity while revealing additional positive and negative associations such as PIK3CA amplification and PTEN mutation, respectively. These patient selection determinants are being assayed in the ongoing NVP-BYL719 clinical trials.

UCSF Small Molecule Discovery Center: innovation, collaboration and chemical biology in the Bay Area.

The Small Molecule Discovery Center (SMDC) at the University of California, San Francisco, works collaboratively with the scientific community to solve challenging problems in chemical biology and drug discovery. The SMDC includes a high throughput screening facility, medicinal chemistry, and research labs focused on fundamental problems in biochemistry and targeted drug delivery. Here, we outline our HTS program and provide examples of chemical tools developed through SMDC collaborations. We have an active research program in developing quantitative cell-based screens for primary cells and whole organisms; here, we describe whole-organism screens to find drugs against parasites that cause neglected tropical diseases. We are also very interested in target-based approaches for so-called "undruggable", protein classes and fragment-based lead discovery. This expertise has led to several pharmaceutical collaborations; additionally, the SMDC works with start-up companies to enable their early-stage research. The SMDC, located in the biotech-focused Mission Bay neighborhood in San Francisco, is a hub for innovative small-molecule discovery research at UCSF.

Pharmacological characterization of the heartbeat in an extant vertebrate ancestor, the Pacific hagfish, Eptatretus stoutii.

Pharmacological ion-channel blockers were used to investigate the spontaneous heart rates in Pacific hagfish, Eptatretus stoutii. Zatebradine, a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker, vastly reduced atrial and ventricular contraction rates in a similar concentration-dependent manner, indicating a major role for HCN in setting intrinsic heart rate. When voltage-gated Na(+) channels were blocked with tetrodotoxin (TTX), atrial contraction rate declined in a dose-dependent manner, but remained faster than ventricular rate even at very high TTX concentrations. This TTX resistance compared with other fish suggests an important role for a TTX-sensitive inactivation-resistant Na(+) current in atrioventricular conduction and chamber synchrony, and a lesser role in setting intrinsic heart rate. T and L-type calcium channel blockers, nickel and nifedipine respectively, also reduced atrial and ventricular contraction rates, nickel having a larger effect on the atrium. These novel results for hagfish are consistent with intrinsic atrial and ventricular rates being set mostly by HCN, with lesser contributions from other ion channels. We suggest that future electrophysiological studies will reveal that hagfishes, with their ancestral position in the evolution of the vertebrate-type chambered heart, share some but not all features of vertebrate intrinsic heart rate control.

A cell-based screen for inhibitors of flagella-driven motility in Chlamydomonas reveals a novel modulator of ciliary length and retrograde actin flow.

Cilia are motile and sensory organelles with critical roles in physiology. Ciliary defects can cause numerous human disease symptoms including polycystic kidneys, hydrocephalus, and retinal degeneration. Despite the importance of these organelles, their assembly and function is not fully understood. The unicellular green alga Chlamydomonas reinhardtii has many advantages as a model system for studies of ciliary assembly and function. Here we describe our initial efforts to build a chemical-biology toolkit to augment the genetic tools available for studying cilia in this organism, with the goal of being able to reversibly perturb ciliary function on a rapid time-scale compared to that available with traditional genetic methods. We screened a set of 5520 compounds from which we identified four candidate compounds with reproducible effects on flagella at nontoxic doses. Three of these compounds resulted in flagellar paralysis and one induced flagellar shortening in a reversible and dose-dependent fashion, accompanied by a reduction in the speed of intraflagellar transport. This latter compound also reduced the length of cilia in mammalian cells, hence we named the compound "ciliabrevin" due to its ability to shorten cilia. This compound also robustly and reversibly inhibited microtubule movement and retrograde actin flow in Drosophila S2 cells. Ciliabrevin may prove especially useful for the study of retrograde actin flow at the leading edge of cells, as it slows the retrograde flow in a tunable dose-dependent fashion until flow completely stops at high concentrations, and these effects are quickly reversed upon washout of the drug.

Obesogenic high fat western diet induces oxidative stress and apoptosis in rat heart.

Feeding Wistar rats a high calorie "Western" diet (45% fat) for up to 48 weeks induces obesity and cardiac dysfunction, while a high fat diet (60% fat) induces obesity only. Here we investigated the molecular "footprints" of the two forms of diet-induced obesity in the heart. In rats fed Western diet for a long term, cardiac mRNA transcript levels of malic enzyme were decreased (-72%, P < 0.05), suggesting impaired anaplerotic flux of the Krebs cycle (KC) and mitochondrial dysfunction. In addition, there was a marked decrease in the expression of the transcription factor MEF2C (myocyte enhancer factor 2C) and its target gene SERCA2a (sarco-endo-plasmic reticulum Ca(2+)-ATPase). Oxidative stress was reflected in reduced transcript levels of manganese superoxide dismutase, glutathione peroxidase 1, and increased protein levels of mitochondrial transcription factor A, suggesting compensatory mitochondrial biogenesis in the face of increased mitochondrial damage. Oxidant injury was accompanied by increased protein glycosylation, increased transcript levels of glutamine fructose 6-phosphate amidotransferase 2, and decreased protein levels of acetyl Co-A carboxylase. Lastly, apoptosis was evident by TUNEL positivity and elevated mRNA transcript levels and activity of caspase 3. Consistent with these results, protein levels of Bcl2 were markedly reduced. We conclude that inadequate supplementation of KC intermediates due to reduced levels of malic enzyme, downregulation of MEF2C and its target gene SERCA2a, oxidative stress, and programmed cell death are all potential contributors to contractile dysfunction of the heart.

An unusual form of fatal ethanol intoxication.

Forensic pathologists are very familiar with deaths due to ethanol intoxication. The overwhelming majority of these deaths are a result of the oral ingestion of ethanol. We report an unusual case of an individual who expired in his secured residence after self administration of a wine enema. Toxicology showed an ethanol concentration of 0.40 g/dL in the blood and 0.41 g/dL in the vitreous fluid. Scene investigation was of paramount importance in determining the unusual method by which the decedent absorbed the alcoholic beverage.

Multiplex GPCR assay in reverse transfection cell microarrays.

G protein-coupled receptors (GPCRs) are a superfamily of proteins that include some of the most important drug targets in the pharmaceutical industry. Despite the success of this group of drugs, there remains a need to identify GPCR-targeted drugs with greater selectivity, to develop screening assays for validated targets, and to identify ligands for orphan receptors. To address these challenges, the authors have created a multiplexed GPCR assay that measures greater than 3000 receptor: ligand interactions in a single microplate. The multiplexed assay is generated by combining reverse transfection in a 96-well plate format with a calcium flux readout. This assay quantitatively measures receptor activation and inhibition and permits the determination of compound potency and selectivity for entire families of GPCRs in parallel. To expand the number of GPCR targets that may be screened in this system, receptors are cotransfected with plasmids encoding a promiscuous G protein, permitting the analysis of receptors that do not normally mobilize intracellular calcium upon activation. The authors demonstrate the utility of reverse transfection cell microarrays to GPCR-targeted drug discovery with examples of ligand selectivity screening against a panel of GPCRs as well as dose-dependent titrations of selected agonists and antagonists.