Miniaturized receptor binding assays: complications arising from ligand depletion.

The advent of miniaturized assay formats has made possible the screening of large numbers of compounds against a single target, known as high-throughput screening. Despite this clear advantage, assay miniaturization also increases the risk of ligand depletion, where the actual concentration of free ligand is significantly lower than that added. This, in turn, complicates the interpretation of data from such assays, potentially introducing significant error if not recognized. In this study, the effects of reducing assay volume on radioligand Kd and competitor Ki values have been investigated, using the muscarinic M(3) receptor as a model system. It was found that assay miniaturization caused dramatic effects, with up to a 30-fold underestimation of ligand affinity. A theoretical model was developed and shown to accurately predict both the degree of ligand depletion in any given assay volume and the effect of this depletion on affinity estimates for competing ligands. Importantly, it was found that in most cases, errors introduced by ligand depletion could be largely corrected for by the use of appropriate analysis methods. In addition to those previously described by others, the authors propose a simple method capable of correcting errors in competition binding experiments performed in conditions of ligand depletion

Mir-210 promotes sensory hair cell formation in the organ of Corti

Background: Hearing loss is the most common sensory defect with several hundred million people worldwide having hearing disorders. In most cases, the cause of hearing loss is related to the degeneration and death of hair cells and their associated spiral ganglion neurons. However, despite this knowledge, relatively few studies have reported regeneration of the auditory system. Significant gaps remain in our understanding of the molecular mechanisms underpinning auditory function, including the factors required for sensory cell regeneration. Recently, the identification of transcriptional activators and repressors of hair cell fate has been augmented by the discovery of microRNAs (miRNAs) associated with hearing loss. As miRNAs are central players of differentiation and cell fate, identification of miRNAs and their gene targets may reveal new pathways for hair cell regeneration and thereby provide new avenues for the treatment of hearing loss. Results: In order to identify new genetic elements enabling regeneration of sensory hair cells in the inner ear, next-generation miRNA sequencing (miRSeq) was used to identify the most prominent microRNAs expressed in the mouse embryonic inner ear cell line UB/OC-1 during differentiation towards a hair cell like phenotype. Based on miRSeq we selected eight most differentially expressed miRNAs for further characterization. Of those, miR-210 knock-down in vitro resulted in hair cell marker expression in UB-OC1, whereas ectopic expression of miR-210 resulted in new hair cell formation in cochlear explants. By using a lineage tracing mouse model, we identified transdifferentiation of supporting epithelial cells as the likely mechanism for new hair cell formation. Potential miR-210 targets were predicted in silico and identified experimentally using a miR-trap approach. Conclusion: MiRSeq followed by ex vivo validation revealed miR-210 as a novel factor driving transdifferentiation of supporting epithelial cells to sensory hair cells. Our data suggest that miR-210 might be a potential new factor for hearing loss therapy. Moreover, identification of inner ear pathways regulated by miR-210 identified novel drug targets for the treatment of hearing loss

Blockade of metallothioneins 1 and 2 increases skeletal muscle mass and strength

Abstract Metallothioneins are proteins that are involved in intracellular zinc storage and transport. Their expression levels have been reported to be elevated in several settings of skeletal muscle atrophy. Here, we demonstrate that these genes are also induced coincident with aging. We therefore investigated the effect of metallothionein blockade on skeletal muscle anabolism in vitro and in vivo, and found that concomitant abrogation of metallothionein 1 and 2 activates the Akt pathway, promotes myotube growth, specifically drives type IIb fiber hypertrophy and ultimately increases muscle strength. Silencing of metallothioneins results in elevated cytosolic zinc; increasing intracellular zinc levels are sufficient to mimic the effects of metallothionein blockade on myotube hypertrophy. We thereby provide direct evidence of the mechanisms by which metallothioneins can modulate skeletal muscle mass. Importantly, the beneficial effects of metallothionein blockade on muscle mass and function are preserved in the presence of a strong catabolic stimulus: treatment with glucocorticoids. Taken together, our results suggest that blockade of metallothioneins constitutes a promising approach for the treatment of muscle diseases

In vitro and in vivo pharmacological characterization of 5-[(R)-2-(5,6-diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one (indacaterol), a novel inhaled beta(2) adrenoceptor agonist with a 24-h duration of action.

Here, we describe the preclinical pharmacological profile of 5-[(R)-2-(5,6-diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one (indacaterol), a novel, chirally pure inhaled beta(2) adrenoceptor agonist, in comparison with marketed drugs. Indacaterol is close to a full agonist at the human beta(2) adrenoceptor (E(max) = 73 +/- 1% of the maximal effect of isoprenaline; pEC(50) = 8.06 +/- 0.02), whereas salmeterol displays only partial efficacy (38 +/- 1%). The functional selectivity profile of indacaterol over beta(1) human adrenoceptors is similar to that of formoterol, whereas its beta(3) adrenoceptor selectivity profile is similar to that of formoterol and salbutamol. In isolated superfused guinea pig trachea, indacaterol has a fast onset of action (30 +/- 4 min) similar to formoterol and salbutamol, and a long duration of action (529 +/- 99 min) comparable with salmeterol. In the conscious guinea pig, when given intratracheally as a dry powder, indacaterol inhibits 5-hydroxytryptamine-induced bronchoconstriction for at least 24 h, whereas salmeterol, formoterol, and salbutamol have durations of action of 12, 4, and 2 h, respectively. When given via nebulization to anesthetized rhesus monkeys, all of the compounds dose-dependently inhibit methacholine-induced bronchoconstriction, although indacaterol produces the most prolonged bronchoprotective effect and induces the lowest increase in heart rate for a similar degree of antibronchoconstrictor activity. In conclusion, the preclinical profile of indacaterol suggests that this compound has a superior duration of action compatible with once-daily dosing in human, together with a fast onset of action and an improved cardiovascular safety profile over marketed inhaled beta(2) adrenoceptor agonists

Additional file 3: of MiR-210 promotes sensory hair cell formation in the organ of corti

Table of miRNA raw counts. Raw counts from sequencing of each of the small miRNA libraries. The number of sequences that correspond to each miRNA is listed. (XLSM 60 kb

Additional file 4: of MiR-210 promotes sensory hair cell formation in the organ of corti

Table of miRNAs normalized in reads-per-million (RPM). The number of reads of each miRNA was normalized by dividing the raw counts by the total number of million aligned reads per sample, i.e. reads per million (RPM). (XLSM 98 kb

Additional file 1: of MiR-210 promotes sensory hair cell formation in the organ of corti

Number of miRSeq reads and alignment statistics. (XLSM 8 kb

The identification of the C3a Receptor (C3AR1) as the target of the VGF derived peptide TLQP-21 in rodent cells

TLQP-21, a peptide derived from VGF (non-acronymic) by proteolytic processing, has been shown to modulate energy metabolism, differentiation and cellular response to stress. Although extensively investigated, the receptor for this endogenous peptide has not previously been described. This report describes the use of a series of studies that show G protein-coupled receptor (GPCR)-mediated biological activity of TLQP-21as well as signalling in CHO-K1 cells. Unbiased genome wide sequencing of the transcriptome of the responsive CHO-K1 cells identified a priority list of possible GPCRs bringing about this activity. Using the biological activity generated by this peptide the possible targets of this ligand were tested by screening a series of defined receptor antagonists as well as siRNAs to inhibit expression of these putative receptors. The results of these studies are all consistent with the receptor of the TLQP-21 peptide in CHO-K1 cells being C3AR1. In addition the sensitivity of TLQP-21 signalling to pertussis toxin is also consistent with what is known about the signalling pathway of this receptor. We further demonstrate that the binding of TLQP-21 to the C3AR1 goes beyond signalling as we show thatTLQP-21 has a migratory role in RAW264.7 mouse cell