Efflux transport of nicotine, cotinine and trans-3 '-hydroxycotinine glucuronides by human hepatic transporters

Nicotine is the addiction causing alkaloid in tobacco, and it is used in smoking cessation therapies. Although the metabolic pathways of nicotine are well known and mainly occur in the liver, the transport of nicotine and its metabolites is poorly characterized. The highly hydrophilic nature and urinary excretion of nicotine glucuronide metabolites indicate that hepatic basolateral efflux transporters mediate their excretion. We aimed here to find the transporters responsible for the hepatic excretion of nicotine, cotinine and trans-3 '-hydroxycotinine (OH-cotinine) glucuronides. To this end, we tested their transport by multidrug resistance-associated proteins 1 (MRP1, ABCC1) and MRP3-6 (ABCC3-6), which are located on the basolateral membranes of hepatocytes, as well as MRP2 (ABCC2), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance protein 1 (MDR1, P-gp, ABCB1) that are expressed in the apical membranes of these cells. ATP-dependent transport of these glucuronides was evaluated in inside-out membrane vesicles expressing the transporter of interest. In addition, potential interactions of both the glucuronides and parent compounds with selected transporters were tested by inhibition assays. Considerable ATP-dependent transport was observed only for OH-cotinine glucuronide by MRP3. The kinetics of this transport activity was characterized, resulting in an estimated K-m value of 895 mu mol/L. No significant transport was found for nicotine or cotinine glucuronides by any of the tested transporters at either 5 or 50 mu mol/L substrate concentration. Furthermore, neither nicotine, cotinine nor OH-cotinine inhibited MRP2-4, BCRP or MDR1. In this study, we directly examined, for the first time, efflux transport of the three hydrophilic nicotine glucuronide metabolites by the major human hepatic efflux transporters. Despite multiple transporters studied here, our results indicate that an unknown transporter may be responsible for the hepatic excretion of nicotine and cotinine glucuronides.Peer reviewe

Transmembrane Domain Single-Nucleotide Polymorphisms Impair Expression and Transport Activity of ABC Transporter ABCG2

To study the function and expression of nine naturally occurring single-nucleotide polymorphisms (G406R, F431L, S441N, P480L, F489L, M515R, L525R, A528T and T542A) that are predicted to reside in the transmembrane regions of the ABC transporter ABCG2. The transport activity of the variants was tested in inside-out membrane vesicles from Sf9 insect and human derived HEK293 cells overexpressing ABCG2. Lucifer Yellow and estrone sulfate were used as probe substrates of activity. The expression levels and cellular localization of the variants was compared to the wild-type ABCG2 by western blotting and immunofluorescence microscopy. All studied variants of ABCG2 displayed markedly decreased transport in both Sf9-ABCG2 and HEK293-ABCG2 vesicles. Impaired transport could be explained for some variants by altered expression levels and cellular localization. Moreover, the destructive effect on transport activity of variants G406R, P480L, M515R and T542A is, to our knowledge, reported for the first time. These results indicate that the transmembrane region of ABCG2 is sensitive to amino acid substitution and that patients harboring these ABCG2 variant forms could suffer from unexpected pharmacokinetic events of ABCG2 substrate drugs or have an increased risk for diseases such as gout where ABCG2 is implicated.Peer reviewe

Food Additives as Inhibitors of Intestinal Drug Transporter OATP2B1

Food additives are compounds that are added to food and beverage to improve the taste, color, preservation, or composition. Generally, food additives are considered safe for human use due to safety evaluations conducted by food safety authorities and high safety margins applied to permitted usage levels. However, the interaction potential of food additives with simultaneously administered medication has not received much attention. Even though many food additives are poorly absorbed into systemic circulation, high concentrations could exist in the intestinal lumen, making intestinal drug transporters, such as the uptake transporter organic anion transporting polypeptide 2B1 (OATP2B1), a possible site of food additive-drug interactions. In the present work, we aimed to characterize the interaction of a selection of 25 food additives including colorants, preservatives, and sweeteners with OATP2B1 in vitro. In human embryonic kidney 293 (HEK293) cells transiently overexpressing OATP2B1 or control, uptake of dibromofluorescein was studied with and without 50 mu M food additive at pH 7.4. As OATP2B1 displays substrate- and pH-dependent transport functions and the intraluminal pH varies along the gastrointestinal tract, we performed the studies also at pH 5.5 using estrone sulfate as an OATP2B1 substrate. Food additives that inhibited OATP2B1-mediated substrate transport by >= 50% were subjected to dose-response studies. Six colorants were identified and validated as OATP2B1 inhibitors at pH 5.5, but only three of these were categorized as inhibitors at pH 7.4. One sweetener was validated as an inhibitor under both assay conditions, whereas none of the preservatives exhibited >= 50% inhibition of OATP2B1-mediated transport. Extrapolation of computed inhibitory constants (K-i values) to estimations of intestinal food additive concentrations implies that selected colorants could inhibit intestinal OATP2B1 also in vivo. These results suggest that food additives, especially colorants, could alter the pharmacokinetics of orally administered OATP2B1 substrate drugs, although further in vivo studies are warranted to understand the overall clinical consequences of the findings.Peer reviewe

Fermi surface induced lattice distortion in NbTe2_2

The origin of the monoclinic distortion and domain formation in the quasi two-dimensional layer compound NbTe$_2$ is investigated. Angle-resolved photoemission shows that the Fermi surface is pseudogapped over large portions of the Brillouin zone. Ab initio calculation of the electron and phonon bandstructure as well as the static RPA susceptibility lead us to conclude that Fermi surface nesting and electron-phonon coupling play a key role in the lowering of the crystal symmetry and in the formation of the charge density wave phase

Spatiotemporal expansion of primary progenitor zones in the developing human cerebellum

We present histological and molecular analyses of the developing human cerebellum from 30 days after conception to 9 months after birth. Differences in developmental patterns between humans and mice include spatiotemporal expansion of both ventricular and rhombic lip primary progenitor zones to include subventricular zones containing basal progenitors. The human rhombic lip persists longer through cerebellar development than in the mouse and undergoes morphological changes to form a progenitor pool in the posterior lobule, which is not seen in other organisms, not even in the nonhuman primate the macaque. Disruptions in human rhombic lip development are associated with posterior cerebellar vermis hypoplasia and Dandy-Walker malformation. The presence of these species-specific neural progenitor populations refines our insight into human cerebellar developmental disorders

UC-Secure CRS Generation for SNARKs

Zero-knowledge SNARKs (zk-SNARKs) have recently found various applications in verifiable computation and blockchain applications (Zerocash), but unfortunately they rely on a common reference string (CRS) that has to be generated by a trusted party. A standard suggestion, pursued by Ben Sasson et al. [IEEE S&P, 2015], is to generate CRS via a multi-party protocol. We enhance their CRS-generation protocol to achieve UC-security. This allows to safely compose the CRS-generation protocol with the zk-SNARK in a black-box manner with the insurance that the security of the zk-SNARK is not influenced. Differently from the previous work, the new CRS-generation protocol also avoids the random oracle model which is typically not required by zk-SNARKs themselves. As a case study, we apply the protocol to the state-of-the-art zk-SNARK by Groth [EUROCRYPT, 2016]

Molecular imaging of cell death in vivo by a novel small molecule probe

Apoptosis has a role in many medical disorders, therefore assessment of apoptosis in vivo can be highly useful for diagnosis, follow-up and evaluation of treatment efficacy. ApoSense is a novel technology, comprising low molecular-weight probes, specifically designed for imaging of cell death in vivo. In the current study we present targeting and imaging of cell death both in vitro and in vivo, utilizing NST-732, a member of the ApoSense family, comprising a fluorophore and a fluorine atom, for both fluorescent and future positron emission tomography (PET) studies using an 18F label, respectively. In vitro, NST-732 manifested selective and rapid accumulation within various cell types undergoing apoptosis. Its uptake was blocked by caspase inhibition, and occurred from the early stages of the apoptotic process, in parallel to binding of Annexin-V, caspase activation and alterations in mitochondrial membrane potential. In vivo, NST-732 manifested selective uptake into cells undergoing cell-death in several clinically-relevant models in rodents: (i) Cell-death induced in lymphoma by irradiation; (ii) Renal ischemia/reperfusion; (iii) Cerebral stroke. Uptake of NST-732 was well-correlated with histopathological assessment of cell-death. NST-732 therefore represents a novel class of small-molecule detectors of apoptosis, with potential useful applications in imaging of the cell death process both in vitro and in vivo