Densitometry data generated for Western blots are commonly used to compare protein abundance between samples. In the last decade, it has become apparent that assumptions underpinning these comparisons are often violated in studies reporting Western blot data in the literature. These violations can lead to erroneous interpretations of data and may contribute to poor reproducibility of research. We assessed the reliability of Western blot data obtained to study human myometrial tissue proteins. We ran dilution series of protein lysates to explore the linearity of densitometry data. Proteins analysed included αSMA, HSP27, ERK1/2, and GAPDH. While ideal densitometry data are directly proportional to protein abundance, our data confirm that densitometry data often deviate from this ideal, in which case they can fit nonproportional linear or hyperbolic mathematical models and can reach saturation. Nonlinear densitometry data were observed when Western blots were detected using infrared fluorescence or chemiluminescence, and under different SDS-PAGE conditions. We confirm that ghosting artefacts associated with overabundance of proteins of interest in Western blots can skew findings. We also confirm that when data to be normalised are not directly proportional to protein abundance, it is a mistake to use the normalisation technique of dividing densitometry data from the protein-of-interest with densitometry data from loading control protein(s), as this can cause the normalised data to be unusable for making comparisons. Using spiked proteins in a way that allowed us to control the total protein amount per lane, while only changing the amount of spiked proteins, we confirm that nonlinearity and saturation of densitometry data, and errors introduced from normalisation processes, can occur in routine assays that compare equal amounts of lysate. These findings apply to all Western blot studies, and we highlight quality control checks that should be performed to make Western blot data more quantitative.

Recent advances in the arena of theranostics have necessitated a re-examining of previously established fields. The existing paradigm of therapeutic thyroid-stimulating hormone receptor (TSHR) targeting in the post-surgical management of differentiated thyroid cancer using levothyroxine and recombinant human thyroid-stimulating hormone (TSH) is well understood. However, in an era of personalized medicine, and with an increasing awareness of the risk profile of longstanding pharmacological hyperthyroidism, it is imperative clinicians understand the molecular basis and magnitude of benefit for individual patients. Furthermore, TSHR has been recently re-conceived as a selective target for residual metastatic thyroid cancer, with pilot data demonstrating effective targeting of nanoparticles to thyroid cancers using this receptor as a target. This review examines the evidence for TSHR signaling as an oncogenic pathway and assesses the evidence for ongoing TSHR expression in thyroid cancer metastases. Priorities for further research are highlighted.

Estrogens are key mediators of increased uterine contractility at labor. We sought to determine whether membrane-associated estrogen receptors, such as the recently described seven-transmembrane receptor G protein-coupled receptor 30 (GPR30), mediated some of this effect. Using human myometrium obtained at term cesarean section before or after the onset of labor, we demonstrated the presence of GPR30 mRNA and protein using quantitative RT-PCR and Western blotting. GPR30 receptor was localized to the cell membrane and often colocalized with calveolin-1. Using the specific estrogen membrane receptor agonist G-1 and myometrial explants, we showed that membrane receptor activation led to phosphorylation of MAPK and the actin-modifying small heat shock protein 27. Using myometrial strips incubated with G-1 or vehicle we demonstrated that estrogen membrane receptor activation increased the myometrial contractile response to oxytocin. These data suggest that activation of the plasma membrane estrogen receptor GPR30 likely participates in the physiology of the human myometrium during pregnancy and identifies it as a potential target to modify uterine activity.

Human ether-a-go-go-related gene (hERG) potassium channels determine cardiac action potential and contraction duration. Human uterine contractions are underpinned by an action potential that also possesses an initial spike followed by prolonged depolarization. Here we show that hERG channel proteins (α-conducting and β-inhibitory subunits) and hERG currents exist in isolated patch-clamped human myometrial cells. We show that hERG channel activity suppresses contraction amplitude and duration before labour, thereby facilitating quiescence. During established labour, expression of β-inhibitory protein is markedly enhanced, resulting in reduced hERG activity that is associated with an increased duration of uterine action potentials and contractions. Thus, changes in hERG channel activity contribute to electrophysiological mechanisms that produce contractions during labour. We also demonstrate that this system fails in women with elevated BMI, who have enhanced hERG activity as a result of low β-inhibitory protein expression, which likely contributes to the weak contractions and poor labour outcomes observed in many obese women necessitating caesarean delivery.