Urinary cell microRNA-based prognostic classifier for non-muscle invasive bladder cancer.

Current prognostic tools for non-muscle invasive bladder cancer (NMIBC) do not have enough discriminative capacity to predict the risk of tumour progression. This study aimed to identify urinary cell microRNAs that may be useful as non-invasive predictive biomarkers of tumour progression in NMIBC patients. To this end, 210 urine samples from NMIBC patients were included in the study. RNA was extracted from urinary cells and expression of 8 microRNAs, previously described by our group, was analysed by quantitative PCR. A tumour progression predicting model was developed by Cox regression analysis and validated by bootstrapping. Regression analysis identified miR-140-5p and miR-92a-3p as independent predictors of tumour progression. The risk score derived from the model containing these two microRNAs was able to discriminate between two groups with a highly significant different probability of tumour progression (HR, 5.204; p<0.001) which was maintained when patients were stratified according to tumour risk. The algorithm was also able to identify two groups with different cancer-specific survival (HR, 3.879; p=0.021). Although the data needs to be externally validated, miRNA analysis in urine appears to be a valuable prognostic tool in NMIBC patients.

Pain relief after triamcinolone infiltration in patients with bladder pain syndrome with Hunner's ulcers.

INTRODUCTION AND HYPOTHESIS: Bladder pain syndrome (BPS) is a chronic condition with severe implications in the patient's quality of life with no definitive treatment. Our objective was to assess pain relief after triamcinolone injection in patients with BPS with Hunner's ulcers (HU). METHODS: Retrospective study of 20 consecutive patients with BPS treated at the Hospital Clinic of Barcelona with triamcinolone injection with flexible cystoscope between 2015 and 2016. Pain was assessed according to the visual analog scale (VAS) (0-10) before and after treatment. Outcomes were compared using Student's t test for paired samples. RESULTS: Twenty-seven procedures were performed in 20 patients, who were followed up for a median of 7 months (range 1-15). Median age was 75 years (52-86), and median time from diagnosis to treatment was 4.5 years (1-7). Fifteen (75 %) patients had received treatment with corticoid injection for BPS before entering the study. Pre- and postreatment VAS was 8 and 2.5 (p < 0.001), respectively. Pre -and postreatment VAS in those with muscular pain was 8 and 5 (p = 0.012), respectively and in those without muscular pain was 8 and 2 (p < 0.001), respectively. Three (15 %) patients required retreatment due to nonresponse and 5 (25 %) patients for pain recurrence after 4 months (3.5-8). Four of them (50 %) were performed with triamcinolone injection again. Seven of ten patients (70 %) followed for ≥8 months required at least one retreatment. CONCLUSION: Triamcinolone injection for HU in patients with BPS is associated with significant pain reduction. However, most patients will require retreatment.

Detergent-induced stabilization and improved 3D map of the human heteromeric amino acid transporter 4F2hc-LAT2.

Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-ß-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level.

Structural bases for the interaction and stabilization of the human amino acid transporter LAT2 with its ancillary protein 4F2hc.

Heteromeric amino acid transporters (HATs) are the unique example, known in all kingdoms of life, of solute transporters composed of two subunits linked by a conserved disulfide bridge. In metazoans, the heavy subunit is responsible for the trafficking of the heterodimer to the plasma membrane, and the light subunit is the transporter. HATs are involved in human pathologies such as amino acidurias, tumor growth and invasion, viral infection and cocaine addiction. However structural information about interactions between the heavy and light subunits of HATs is scarce. In this work, transmission electron microscopy and single-particle analysis of purified human 4F2hc/L-type amino acid transporter 2 (LAT2) heterodimers overexpressed in the yeast Pichia pastoris, together with docking analysis and crosslinking experiments, reveal that the extracellular domain of 4F2hc interacts with LAT2, almost completely covering the extracellular face of the transporter. 4F2hc increases the stability of the light subunit LAT2 in detergent-solubilized Pichia membranes, allowing functional reconstitution of the heterodimer into proteoliposomes. Moreover, the extracellular domain of 4F2hc suffices to stabilize solubilized LAT2. The interaction of 4F2hc with LAT2 gives insights into the structural bases for light subunit recognition and the stabilizing role of the ancillary protein in HATs.

Expression of human heteromeric amino acid transporters in the yeast Pichia pastoris.

Human heteromeric amino acid transporters (HATs) play key roles in renal and intestinal re-absorption, cell redox balance and tumor growth. These transporters are composed of a heavy and a light subunit, which are connected by a disulphide bridge. Heavy subunits are the two type II membrane N-glycoproteins rBAT and 4F2hc, while L-type amino acid transporters (LATs) are the light and catalytic subunits of HATs. We tested the expression of human 4F2hc and rBAT as well as seven light subunits in the methylotrophic yeast Pichia pastoris. 4F2hc and the light subunit LAT2 showed the highest expression levels and yields after detergent solubilization. Co-transformation of both subunits in Pichia cells resulted in overexpression of the disulphide bridge-linked 4F2hc/LAT2 heterodimer. Two sequential affinity chromatography steps were applied to purify detergent-solubilized heterodimers yielding ~1mg of HAT from 2l of cell culture. Our results indicate that P. pastoris is a convenient system for the expression and purification of human 4F2hc/LAT2 for structural studies.