Lanthanum-fluoride electrode-based methods to monitor fluoride transport in cells and reconstituted lipid vesicles.

Fluoride (F-) export proteins, including F- channels and F- transporters, are widespread in biology. They contribute to cellular resistance against fluoride ion, which has relevance as an ancient xenobiotic, and in more modern contexts like organofluorine biosynthesis and degradation or dental medicine. This chapter summarizes quantitative methods to measure fluoride transport across membranes using fluoride-specific lanthanum-fluoride electrodes. Electrode-based measurements can be used to measure unitary fluoride transport rates by membrane proteins that have been purified and reconstituted into lipid vesicles, or to monitor fluoride efflux into living microbial cells. Thus, fluoride electrode-based measurements yield quantitative mechanistic insight into one of the major determinants of fluoride resistance in microorganisms, fungi, yeasts, and plants.

SVCT2/SLC23A2 is a sodium-dependent urate transporter: functional properties and practical application.

Urate transporters play a pivotal role in urate handling in the human body, but the urate transporters identified to date do not account for all known molecular processes of urate handling, suggesting the presence of latent machineries. We recently showed that a urate transporter SLC2A12 is also a physiologically important exporter of ascorbate (the main form of vitamin C in the body) that would cooperate with an ascorbate importer, sodium-dependent vitamin C transporter 2 (SVCT2). Based on the dual functions of SLC2A12 and cooperativity between SLC2A12 and SVCT2, we hypothesized that SVCT2 might be able to transport urate. To test this proposal, we conducted cell-based analyses using SVCT2-expressing mammalian cells. The results demonstrated that SVCT2 is a novel urate transporter. Vitamin C inhibited SVCT2-mediated urate transport with a half-maximal inhibitory concentration of 36.59 µM, suggesting that the urate transport activity may be sensitive to physiological ascorbate levels in blood. Similar results were obtained for mouse Svct2. Further, using SVCT2 as a sodium-dependent urate importer, we established a cell-based urate efflux assay that will be useful for identification of other novel urate exporters as well as functional characterization of nonsynonymous variants of already-identified urate exporters including ATP-binding cassette transporter G2. While more studies will be needed to elucidate the physiological impact of SVCT2-mediated urate transport, our findings deepen understanding of urate transport machineries.

Measuring Efflux and Permeability in Mycobacteria.

Mycobacteria are intrinsically resistant to most antimicrobials, which is generally attributed to the impermeability of their cell wall that considerably limits drug uptake. Moreover, like in other pathogenic bacteria, active efflux systems have been widely characterized from diverse mycobacterial species in laboratory conditions, showing that they can promote resistance by extruding noxious compounds prior to their reaching their intended targets. Therefore, the intracellular concentration of a given compound is determined by the balance between permeability, influx, and efflux.Given the urgent need to discover and develop novel antimycobacterial compounds in order to design effective therapeutic strategies, the contributions to drug resistance made by the controlled permeability of the cell wall and the increased activity of efflux pumps must be determined. In this chapter, we will describe a method that allows (1) the measuring of permeability and the quantification of general efflux activity of mycobacteria, by the study of the transport (influx and efflux) of fluorescent compounds, such as ethidium bromide; and (2) the screening of compounds in search of agents that increase the permeability of the cell wall and efflux inhibitors that could restore the effectiveness of antimicrobials that are subject to efflux.

Novel DiOC3 96-well real-time efflux assay for discovery of NorA efflux pump inhibitors in Staphylococcus aureus.

The NorA efflux pump is one of the most studied efflux systems in Staphylococcus aureus and confers multidrug resistance to a variety of dyes and antimicrobial compounds. Hence, inhibition of the NorA efflux pump might be a viable option for restoring susceptibility to antibiotics like fluoroquinolones. Fluorescent real-time efflux assays are important tools to identify putative efflux pump inhibitors. Nevertheless, the number of available compounds for usage in Staphylococcus aureus is limited. Previously, a 3-dipropyloxacarbocyanine iodide (DiOC3) efflux assay was published that circumvented problems associated with the usage of ethidium bromide, namely slow efflux and suggested mutagenicity. However, the DiOC3 assay protocol was cuvette - based and therefore needs to be adapted to the 96-well plate format. Hence, we optimized this assay for usage with 96-well plates. The new assay allows for rapid high-throughput efflux pump inhibitor screening.

Potent Synergistic Interactions between Lopinavir and Azole Antifungal Drugs against Emerging Multidrug-Resistant Candida auris.

The limited therapeutic options and the recent emergence of multidrug-resistant Candida species present a significant challenge to human medicine and underscore the need for novel therapeutic approaches. Drug repurposing appears as a promising tool to augment the activity of current azole antifungals, especially against multidrug-resistant Candida auris In this study, we evaluated the fluconazole chemosensitization activities of 1,547 FDA-approved drugs and clinical molecules against azole-resistant C. auris This led to the discovery that lopinavir, an HIV protease inhibitor, is a potent agent capable of sensitizing C. auris to the effect of azole antifungals. At a therapeutically achievable concentration, lopinavir exhibited potent synergistic interactions with azole drugs, particularly with itraconazole against C. auris (fractional inhibitory concentration index [ΣFICI] ranged from 0.04 to 0.09). Additionally, the lopinavir/itraconazole combination enhanced the survival rate of C. auris-infected Caenorhabditis elegans by 90% and reduced the fungal burden in infected nematodes by 88.5% (P < 0.05) relative to that of the untreated control. Furthermore, lopinavir enhanced the antifungal activity of itraconazole against other medically important Candida species, including C. albicans, C. tropicalis, C. krusei, and C. parapsilosis Comparative transcriptomic profiling and mechanistic studies revealed that lopinavir was able to significantly interfere with the glucose permeation and ATP synthesis. This compromised the efflux ability of C. auris and consequently enhanced the susceptibility to azole drugs, as demonstrated by Nile red efflux assays. Altogether, these findings present lopinavir as a novel, potent, and broad-spectrum azole-chemosensitizing agent that warrants further investigation against recalcitrant Candida infections.

Inhibitory effects of neurotoxin ß-N-methylamino-L-alanine on fertilization and early development of the sea urchin Lytechinus pictus.

Neurotoxin ß-N-methylamino-L-alanine (BMAA) has been widely detected in diverse aquatic organisms and hypothesized as an environmental risk to neurodegenerative diseases in humans. However, the knowledge of its toxicity to marine organisms requires attention. In the present study, embryos and sperm of the sea urchin, Lytechinus pictus, were used to assess the toxicity of BMAA. Effects of BMAA on fertilization and development of sea urchin embryos were measured, and its impacts on efflux transport of sea urchin blastula were also assayed. Results demonstrated that the fertilization and development of embryos were significantly inhibited by high concentrations of BMAA above 300 µg L-1. The EC50 values indicated by active swimming larvae and total larvae numbers at 96 HPF (hours post fertilization) were 165 µg L-1 (1.4 µmol L-1) and 329 µg L-1 (2.8 µmol L-1), respectively. Additionally, sperm exposed to BMAA for 10 min significantly reduced the fertilization ratio of sea urchin eggs. However, the ABC transport activity on the cytomembrane of sea urchin blastula was not inhibited by the presence of BMAA at 50 µg L-1, even up to 500 µg L-1. Abnormal division and developmental malformations occurred at different developmental stages for sea urchin embryos exposed to BMAA at 500 µg L-1. The inhibitory effects of BMAA on sea urchin embryos were reported at the first time in this study, for which the toxicological mechanisms will be explored in future studies.

Intestinal breast cancer resistance protein (BCRP) requires Janus kinase 3 activity for drug efflux and barrier functions in obesity.

Breast cancer resistance protein (BCRP) is a member of ATP-binding cassette (ABC) transporter proteins whose primary function is to efflux substrates bound to the plasma membrane. Impaired intestinal barrier functions play a major role in chronic low-grade inflammation (CLGI)-associated obesity, but the regulation of BCRP during obesity and its role in maintaining the intestinal barrier function during CLGI-associated obesity are unknown. In the present study, using several approaches, including efflux assays, immunoprecipitation, immunoblotting, immunohistochemistry, paracellular permeability assay, FACS, cytokine assay, and immunofluorescence microscopy, we report that obese individuals have compromised intestinal BCRP functions and that diet-induced obese mice recapitulate these outcomes. We demonstrate that the compromised BCRP functions during obesity are because of loss of Janus kinase 3 (JAK3)-mediated tyrosine phosphorylation of BCRP. Our results indicate that JAK3-mediated phosphorylation of BCRP promotes its interactions with membrane-localized ß-catenin essential not only for BCRP expression and surface localization, but also for the maintenance of BCRP-mediated intestinal drug efflux and barrier functions. We observed that reduced intestinal JAK3 expression during human obesity or JAK3 knockout in mouse or siRNA-mediated ß-catenin knockdown in human intestinal epithelial cells all result in significant loss of intestinal BCRP expression and compromised colonic drug efflux and barrier functions. Our results uncover a mechanism of BCRP-mediated intestinal drug efflux and barrier functions and establish a role for BCRP in preventing CLGI-associated obesity both in humans and in mice.

Synthesis, in silico and in vitro studies of new 1,4-dihydropiridine derivatives for antitumor and P-glycoprotein inhibitory activity.

P-glycoprotein (P-gp) is one of the cell membrane pumps which mediate the efflux of molecules such as anticancer drugs to the extracellular matrix of tumor cells. P_gp is a member of the ATP-binding cassette (ABC) transporter family that is implicated in cancer multidrug resistance (MDR). Since MDR is a contributor to cancer chemotherapy failure, modulation of efflux pumps is a viable therapeutic strategy. In this study, new synthetic 1,4 dihydropiridine (DHP) derivatives containing thiophenyl substitution were tested as inhibitors of P-gp. Efflux assay was conducted to evaluate the intracellular accumulation of Rhodamine123 (Rh123) as a pump substrate. MTT assay, cell cycle analysis and in silico methods were also examined. Flow cytometric analysis revealed that synthetic DHP derivatives (15 µM) increased intracellular concentration of the substrate by 2-3 folds compared with verapamil as a standard P-gp inhibitor. MTT assay on EPG85-257P and its drug-resistant EPG85-257RDB cell line revealed antitumor effects (30-45%) for new DHP derivatives at 15 µM following 72 h incubation. However, MTT test on normal cell line showed negligible toxic effects. Finally combination of synthetic derivatives with doxorubicin showed that these compounds decrease IC50 of doxorubicin in resistant cell lines from 9 to 1.5 µM. Sub-G1 peak-related apoptotic cells showed a stronger effect of synthetic compounds at 5 µM compared with verapamil. Molecular dynamic results showed a high binding affinity between DHP derivative and protein at drug binding site. Findings of these biological tests indicated the antitumor activity and P-gp inhibitory effects of new 1,4-DHP derivatives.

Optimized efflux assay for the NorA multidrug efflux pump in Staphylococcus aureus.

Real-time fluorescent efflux assays are commonly used for measuring the efflux of bacterial pumps. Here we describe an optimized protocol for the NorA efflux pump in S. aureus using DiOC3 instead of ethidium bromide. Glucose and sodium formate were tested as energy carriers. This novel method is fast and reproducible.

Modeling Cystic Fibrosis Using Pluripotent Stem Cell-Derived Human Pancreatic Ductal Epithelial Cells.

UNLABELLED: We established an efficient strategy to direct human pluripotent stem cells, including human embryonic stem cells (hESCs) and an induced pluripotent stem cell (iPSC) line derived from patients with cystic fibrosis, to differentiate into pancreatic ductal epithelial cells (PDECs). After purification, more than 98% of hESC-derived PDECs expressed functional cystic fibrosis transmembrane conductance regulator (CFTR) protein. In addition, iPSC lines were derived from a patient with CF carrying compound frameshift and mRNA splicing mutations and were differentiated to PDECs. PDECs derived from Weill Cornell cystic fibrosis (WCCF)-iPSCs showed defective expression of mature CFTR protein and impaired chloride ion channel activity, recapitulating functional defects of patients with CF at the cellular level. These studies provide a new methodology to derive pure PDECs expressing CFTR and establish a "disease in a dish" platform to identify drug candidates to rescue the pancreatic defects of patients with CF. SIGNIFICANCE: An efficient strategy was established to direct human pluripotent stem cells, including human embryonic stem cells (hESCs) and an induced pluripotent stem cell line derived from patients with cystic fibrosis (CF-iPSCs), to differentiate into pancreatic ductal epithelial cells (PDECs). After purification, more than 98% of hESC-PDECs derived from CF-iPSCs showed defective expression of mature cystic fibrosis transmembrane conductance regulator (CFTR) protein and impaired chloride ion channel activity, recapitulating functional pancreatic defects of patients with CF at the cellular level. These studies provide a new methodology for deriving pure PDECs expressing CFTR, and they establish a "disease-in-a-dish" platform for identifying drug candidates to rescue the pancreatic defects of these patients.

MRP1 and P-glycoprotein expression assays would be useful in the additional detection of treatment non-responders in CML patients without ABL1 mutation.

We evaluated the ability of the rhodamine-123 efflux assay, multidrug resistance-associated protein-1 (MRP1) expression assay and P-glycoprotein (Pgp) expression assay to discriminate chronic myelogenous leukemia (CML) patients who had failed treatment or were at risk of failure. Each assay was performed in blood samples from CML patients (n=224) treated with tyrosine kinase inhibitors, taken at diagnosis (n=14) and follow-up (n=210). Patient samples were categorized as optimal response (n=120), suboptimal response (n=54), and treatment failure (n=36). Treatment-failed patients had a significantly higher MRP1 expression (5.24% vs. 3.54%, P=0.006) and Pgp expression (5.25% vs. 3.48%, P=0.005) than responders. Both MRP1 (%) and Pgp (%) were highly specific (95.2% and 94.5%) and relatively accurate (83.0% and 82.5%) in the detection of treatment non-responders. Of treatment-failed patients, 41.2% had a positive result in at least one assay and of these patients without ABL1 kinase domain mutation, 51.9% were positive in at least one assay. However, the rhodamine-123 efflux assay failed to discriminate two patient groups. Thus, both MRP1 and Pgp expression assays could be useful for additional identification of treatment non-responders in CML patients without ABL1 mutation.

Flow cytometric assessment of P-glycoprotein and multidrug resistance-associated protein activity and expression in canine lymphoma.

AIM: To investigate the activity and expression of P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRPs) in biopsy samples from lymph nodes of 22 dogs at the time of lymphoma diagnosis. MATERIALS AND METHODS: Protein activity and expression were investigated by flow cytometry. Expression was assessed with monoclonal antibodies (C494 for P-gp and MRPm6 for MRP1). P-gp activity was determined by rhodamine 123 (Rho 123) efflux inhibited by verapamil and MRP by 5(6) carboxyfluorescein diacetate (CFDA) efflux inhibited by probenecid. Cell lineage was determined based on the expression of CD79α, CD21, CD3, CD4 and CD8. RESULTS: P-gp and MRP1 were expressed in all samples. Relative activity was low for both transporters and no correlation was found between transporter activity and expression. The majority of lymphomas were of B-cell origin. CONCLUSION: Low transporter activity and lack of correlation with expression suggest that spontaneous up-regulation of P-gp or MRP is not a common phenomenon in canine lymphoma.

Campylobacter jejuni infection suppressed Cl⁻ secretion induced by CFTR activation in T-84 cells.

Campylobacter jejuni causes foodborne disease associated with abdominal pain, gastroenteritis, and diarrhea. These symptoms are induced by bacterial adherence and invasion of host epithelial cells. C. jejuni infection can occur with a low infective dose, suggesting that C. jejuni may have evolved strategies to cope with the bacterial clearance system in the gastrointestinal tract. The mucosa layer is the first line of defense against bacteria. Mucus conditions are maintained by water and anion (especially Cl(-)) movement. Cystic fibrosis transmembrane conductance regulator (CFTR) is the main Cl(-) channel transporting Cl(-) to the lumen. Mutations in CFTR result in dehydrated secreted mucus and bacterial accumulation in the lungs, and recent studies suggest that closely related pathogenic bacteria also may survive in the intestine. However, the relationship between C. jejuni infection and CFTR has been little studied. Here, we used an (125)I(-) efflux assay and measurement of short-circuit current to measure Cl(-) secretion in C. jejuni-infected T-84 human intestinal epithelial cells. The basic state of Cl(-) secretion was unchanged by C. jejuni infection, but CFTR activator was observed to induce Cl(-) secretion suppressed in C. jejuni-infected T-84 cells. The suppression of activated Cl(-) secretion was bacterial dose-dependent and duration-dependent. A similar result was observed during infection with other C. jejuni strains. The mechanism of suppression may occur by affecting water movement or mucus condition in the intestinal tract. A failure of mucus barrier function may promote bacterial adhesion or invasion of host intestinal epithelial cells, thereby causing bacterial preservation in the host intestinal tract.

Lymphocyte P-glycoprotein variability in healthy individuals / Variabilidad de la glicoproteína P linfocitaria en una población sana

The aim of the present work was to describe the distribution of lymphocyte P-glycoprotein activity on a population of healthy individuals, taking also into account sex and age. P-glycoprotein activity in lymphocytes was measured by the Rhodamine 123 efflux assay using flow cytometry, in the presence and absence of verapamil, a P-glycoprotein inhibitor. We obtained a range of P-glycoprotein activity from 1.04 to 3.79. The distribution of the activity in the population studied was better described by a bimodal model, according with the Kolmogorov-Smirnov test. The frequency adjusted to the following equation: F = 0.70 N (2.11; 0.43) + 0.30 N(3.29; 0.26), in which 0.70 and 0.30 represented the proportion of each group, and 0.43 and 0.26 were the standard deviations of the activity of each group, respectively. The study of the relationship between subjects´ age and P-glycoprotein activity showed no statistical significance. When healthy volunteers were separated according to sex, similar distributions were observed, although for men an increase in proportion of higher P-glycoprotein function group was observed. The variability observed in the population studied was important, with some volunteers with very scarce activity and some with a fourfold higher activity. Characterization of P-glycoprotein functionality in the population represents a useful contribution to the beginning of pharmacological treatments that consider its effect on pharmacokinetics and pharmacodynamics of individualized patients.

El objetivo del presente trabajo fue describir la distribución de la actividad de la glicoproteína P linfocitaria en una población de individuos sanos, considerando a su vez el sexo y la edad. La funcionalidad de la glicoproteína P fue determinada mediante el ensayo de eflujo de Rodamina 123, en presencia y ausencia de verapamilo, un inhibidor competitivo de este transportador, determinando la fluorescencia intracelular remanente mediante citometría de flujo. Obtuvimos un rango de actividades de entre 1.04 y 3.79. La distribución de la actividad en la población evaluada se ajusta a un modelo bimodal, según el test de Kolmogorov-Smirnov. La frecuencia ajusta a la siguiente ecuación: F = 0.70 N (2.11; 0.43) + 0.30 N (3.29; 0.26) donde 0.70 y 0.30 representan las proporciones de cada grupo, mientras que 0.43 y 0.26 corresponden al desvío estándar de la actividad de cada grupo respectivamente. Al estudiar la correlación entre la edad de los sujetos y la función de la proteína, no se observaron diferencias significativas. Cuando los individuos fueron clasificados en función del sexo, las distribuciones obtenidas fueron semejantes, aunque para los varones se observó un aumento en la proporción de individuos con alta actividad. La variabilidad observada fue importante, comprendiendo individuos con escasa actividad y otros que presentaron una actividad hasta cuatro veces mayor. La caracterización de la función de la glicoproteína P en la población representa una contribución indispensable para el desarrollo de tratamientos farmacológicos personalizados que consideren el efecto de dicho transportador en la farmacocinética y farmacodinámica de cada paciente.