Visualization of Glutamine Transporter Activities in Living Cells Using Genetically Encoded Glutamine Sensors

Glutamine plays a central role in the metabolism of critical biological molecules such as amino acids, proteins, neurotransmitters, and glutathione. Since glutamine metabolism is regulated through multiple enzymes and transporters, the cellular glutamine concentration is expected to be temporally dynamic. Moreover, differentiation in glutamine metabolism between cell types in the same tissue (e.g. neuronal and glial cells) is often crucial for the proper function of the tissue as a whole, yet assessing cell-type specific activities of transporters and enzymes in such heterogenic tissue by physical fractionation is extremely challenging. Therefore, a method of reporting glutamine dynamics at the cellular level is highly desirable. Genetically encoded sensors can be targeted to a specific cell type, hence addressing this knowledge gap. Here we report the development of Föster Resonance Energy Transfer (FRET) glutamine sensors based on improved cyan and yellow fluorescent proteins, monomeric Teal Fluorescent Protein (mTFP)1 and venus. These sensors were found to be specific to glutamine, and stable to pH-changes within a physiological range. Using cos7 cells expressing the human glutamine transporter ASCT2 as a model, we demonstrate that the properties of the glutamine transporter can easily be analyzed with these sensors. The range of glutamine concentration change in a given cell can also be estimated using sensors with different affinities. Moreover, the mTFP1-venus FRET pair can be duplexed with another FRET pair, mAmetrine and tdTomato, opening up the possibility for real-time imaging of another molecule. These novel glutamine sensors will be useful tools to analyze specificities of glutamine metabolism at the single-cell level

Molecular Marker for Predicting Treatment Response in Advanced Renal Cell Carcinoma: Does the Promise Fulfill Clinical Need?

Renal cell carcinoma (RCC) is largely diagnosed incidentally on imaging taken for unrelated reasons. The management of localized lesions is primarily extirpative with excellent results. Treatment of advanced RCC has evolved over recent years with the use of targeted therapies such as tyrosine kinase inhibitors, mammalian target of rapamycin inhibitors, and antibody-mediated therapies. The treatment response to these targeted therapies is highly variable, with no clear clinical method of identifying patients who will benefit from or not tolerate therapy. The field of molecular markers has evolved significantly in the last decade, with a multitude of markers identified that predict treatment response and drug toxicity. The following review critically evaluates those molecular markers that have been assessed for their utility in predicting treatment response in patients with advanced/metastatic renal cell carcinoma (mRCC). Identifying the ideal treatment for these patients will improve responses to therapy, minimize morbidity, and save significant healthcare dollars

Renal tract malformations: perspectives for nephrologists.

Contains fulltext : 71176.pdf (publisher's version ) (Closed access)Renal tract malformations are congenital anomalies of the kidneys and/or lower urinary tract. One challenging feature of these conditions is that they can present not only prenatally but also in childhood or adulthood. The most severe types of malformations, such as bilateral renal agenesis or dysplasia, although rare, lead to renal failure. With advances in dialysis and transplantation for young children, it is now possible to prevent the early death of at least some individuals with severe malformations. Other renal tract malformations, such as congenital pelviureteric junction obstruction and primary vesicoureteric reflux, are relatively common. Renal tract malformations are, collectively, the major cause of childhood end-stage renal disease. Their contribution to the number of adults on renal replacement therapy is less clear and has possibly been underestimated. Renal tract malformations can be familial, and specific mutations of genes involved in renal tract development can sometimes be found in affected individuals. These features provide information about the causes of malformations but also raise questions about whether to screen relatives. Whether prenatal decompression of obstructed renal tracts, or postnatal initiation of therapies such as prophylactic antibiotics or angiotensin blockade, improve long-term renal outcomes remains unclear