Evaluation of uptake and transport of cationic and anionic ultrasmall iron oxide nanoparticles by human colon cells

Nanoparticles (NPs) are in clinical use or under development for therapeutic imaging and drug delivery. However, relatively little information exists concerning the uptake and transport of NPs across human colon cell layers, or their potential to invade three-dimensional models of human colon cells that better mimic the tissue structures of normal and tumoral colon. In order to gain such information, the interactions of biocompatible ultrasmall superparamagnetic iron oxide nanoparticles (USPIO NPs) (iron oxide core 9-10 nm) coated with either cationic polyvinylamine (aminoPVA) or anionic oleic acid with human HT-29 and Caco-2 colon cells was determined. The uptake of the cationic USPIO NPs was much higher than the uptake of the anionic USPIO NPs. The intracellular localization of aminoPVA USPIO NPs was confirmed in HT-29 cells by transmission electron microscopy that detected the iron oxide core. AminoPVA USPIO NPs invaded three-dimensional spheroids of both HT-29 and Caco-2 cells, whereas oleic acid-coated USPIO NPs could only invade Caco-2 spheroids. Neither cationic aminoPVA USPIO NPs nor anionic oleic acid-coated USPIO NPs were transported at detectable levels across the tight CacoReady? intestinal barrier model or the more permeable mucus-secreting CacoGoblet? model.

The policy relevance of wear emissions from road transport, now and in the future : an international workshop report and consensus statement

Road transport emissions are a major contributor to ambient particulate matter concentrations and have been associated with adverse health effects. Therefore, these emissions are targeted through increasingly stringent European emission standards. These policies succeed in reducing exhaust emissions, but do not address "nonexhaust" emissions from brake wear, tire wear, road wear, and suspension in air of road dust. Is this a problem? To what extent do nonexhaust emissions contribute to ambient concentrations of PM10 or PM2.5? In the near future, wear emissions may dominate the remaining traffic-related PM10 emissions in Europe, mostly due to the steep decrease in PM exhaust emissions. This underlines the need to determine the relevance of the wear emissions as a contribution to the existing ambient PM concentrations, and the need to assess the health risks related to wear particles, which has not yet received much attention. During a workshop in 2011, available knowledge was reported and evaluated so as to draw conclusions on the relevance of traffic-related wear emissions for air quality policy development. On the basis of available evidence, which is briefly presented in this paper, it was concluded that nonexhaust emissions and in particular suspension in air of road dust are major contributors to exceedances at street locations of the PM10 air quality standards in various European cities. Furthermore, wear-related PM emissions that contain high concentrations of metals may (despite their limited contribution to the mass of nonexhaust emissions) cause significant health risks for the population, especially those living near intensely trafficked locations. To quantify the existing health risks, targeted research is required on wear emissions, their dispersion in urban areas, population exposure, and its effects on health. Such information will be crucial for environmental policymakers as an input for discussions on the need to develop control strategies.

The extended GLUT-family of sugar/polyol transport facilitators: nomenclature, sequence characteristics, and potential function of its novel members (review).

During the last 2 years, several novel genes that encode glucose transporter-like proteins have been identified and characterized. Because of their sequence similarity with GLUT1, these genes appear to belong to the family of solute carriers 2A (SLC2A, protein symbol GLUT). Sequence comparisons of all 13 family members allow the definition of characteristic sugar/polyol transporter signatures: (1) the presence of 12 membrane-spanning helices, (2) seven conserved glycine residues in the helices, (3) several basic and acidic residues at the intracellular surface of the proteins, (4) two conserved tryptophan residues, and (5) two conserved tyrosine residues. On the basis of sequence similarities and characteristic elements, the extended GLUT family can be divided into three subfamilies, namely class I (the previously known glucose transporters GLUT1-4), class II (the previously known fructose transporter GLUT5, the GLUT7, GLUT9 and GLUT11), and class III (GLUT6, 8, 10, 12, and the myo-inositol transporter HMIT1). Functional characteristics have been reported for some of the novel GLUTs. Like GLUT1-4, they exhibit a tissue/cell-specific expression (GLUT6, leukocytes, brain; GLUT8, testis, blastocysts, brain, muscle, adipocytes; GLUT9, liver, kidney; GLUT10, liver, pancreas; GLUT11, heart, skeletal muscle). GLUT6 and GLUT8 appear to be regulated by sub-cellular redistribution, because they are targeted to intra-cellular compartments by dileucine motifs in a dynamin dependent manner. Sugar transport has been reported for GLUT6, 8, and 11; HMIT1 has been shown to be a H+/myo-inositol co-transporter. Thus, the members of the extended GLUT family exhibit a surprisingly diverse substrate specificity, and the definition of sequence elements determining this substrate specificity will require a full functional characterization of all members.

D6PK AGCVIII Kinases Are Required for Auxin Transport and Phototropic Hypocotyl Bending in Arabidopsis.

Phototropic hypocotyl bending in response to blue light excitation is an important adaptive process that helps plants to optimize their exposure to light. In Arabidopsis thaliana, phototropic hypocotyl bending is initiated by the blue light receptors and protein kinases phototropin1 (phot1) and phot2. Phototropic responses also require auxin transport and were shown to be partially compromised in mutants of the PIN-FORMED (PIN) auxin efflux facilitators. We previously described the D6 PROTEIN KINASE (D6PK) subfamily of AGCVIII kinases, which we proposed to directly regulate PIN-mediated auxin transport. Here, we show that phototropic hypocotyl bending is strongly dependent on the activity of D6PKs and the PIN proteins PIN3, PIN4, and PIN7. While early blue light and phot-dependent signaling events are not affected by the loss of D6PKs, we detect a gradual loss of PIN3 phosphorylation in d6pk mutants of increasing complexity that is most severe in the d6pk d6pkl1 d6pkl2 d6pkl3 quadruple mutant. This is accompanied by a reduction of basipetal auxin transport in the hypocotyls of d6pk as well as in pin mutants. Based on our data, we propose that D6PK-dependent PIN regulation promotes auxin transport and that auxin transport in the hypocotyl is a prerequisite for phot1-dependent hypocotyl bending.

Mortality among 24,865 workers exposed to polychlorinated biphenyls (PCBs) in three electrical capacitor manufacturing plants: a ten-year update.

The objective of this analysis was to evaluate mortality among a cohort of 24,865 capacitor-manufacturing workers exposed to polychlorinated biphenyls (PCBs) at plants in Indiana, Massachusetts, and New York and followed for mortality through 2008. Cumulative PCB exposure was estimated using plant-specific job-exposure matrices. External comparisons to US and state-specific populations used standardized mortality ratios, adjusted for gender, race, age and calendar year. Among long-term workers employed 3 months or longer, within-cohort comparisons used standardized rate ratios and multivariable Poisson regression modeling. Through 2008, more than one million person-years at risk and 8749 deaths were accrued. Among long-term employees, all-cause and all-cancer mortality were not elevated; of the a priori outcomes assessed only melanoma mortality was elevated. Mortality was elevated for some outcomes of a priori interest among subgroups of long-term workers: all cancer, intestinal cancer and amyotrophic lateral sclerosis (women); melanoma (men); melanoma and brain and nervous system cancer (Indiana plant); and melanoma and multiple myeloma (New York plant). Standardized rates of stomach and uterine cancer and multiple myeloma mortality increased with estimated cumulative PCB exposure. Poisson regression modeling showed significant associations with estimated cumulative PCB exposure for prostate and stomach cancer mortality. For other outcomes of a priori interest--rectal, liver, ovarian, breast, and thyroid cancer, non-Hodgkin lymphoma, Alzheimer disease, and Parkinson disease--neither elevated mortality nor positive associations with PCB exposure were observed. Associations between estimated cumulative PCB exposure and stomach, uterine, and prostate cancer and myeloma mortality confirmed our previous positive findings.

Creatine and guanidinoacetate transport at blood-brain and blood-cerebrospinal fluid barriers.

While it was thought that most of cerebral creatine is of peripheral origin, AGAT and GAMT are well expressed in CNS where brain cells synthesize creatine. While the creatine transporter SLC6A8 is expressed by microcapillary endothelial cells (MCEC) at blood-brain barrier (BBB), it is absent from their surrounding astrocytes. This raised the concept that BBB has a limited permeability for peripheral creatine, and that the brain supplies a part of its creatine by endogenous synthesis. This review brings together the latest data on creatine and guanidinoacetate transport through BBB and blood-CSF barrier (BCSFB) with the clinical evidence of AGAT-, GAMT- and SLC6A8-deficient patients, in order to delineate a clearer view on the roles of BBB and BCSFB in the transport of creatine and guanidinoacetate between periphery and CNS, and on brain synthesis and transport of creatine. It shows that in physiological conditions, creatine is taken up by CNS from periphery through SLC6A8 at BBB, but in limited amounts, and that CNS also needs its own creatine synthesis. No uptake of guanidinoacetate from periphery occurs at BBB except under GAMT deficiency, but a net exit of guanidinoacetate seems to occur from CSF to blood at BCSFB, predominantly through the taurine transporter TauT.

Learning-induced plasticity in auditory spatial representations revealed by electrical neuroimaging.

Auditory spatial representations are likely encoded at a population level within human auditory cortices. We investigated learning-induced plasticity of spatial discrimination in healthy subjects using auditory-evoked potentials (AEPs) and electrical neuroimaging analyses. Stimuli were 100 ms white-noise bursts lateralized with varying interaural time differences. In three experiments, plasticity was induced with 40 min of discrimination training. During training, accuracy significantly improved from near-chance levels to approximately 75%. Before and after training, AEPs were recorded to stimuli presented passively with a more medial sound lateralization outnumbering a more lateral one (7:1). In experiment 1, the same lateralizations were used for training and AEP sessions. Significant AEP modulations to the different lateralizations were evident only after training, indicative of a learning-induced mismatch negativity (MMN). More precisely, this MMN at 195-250 ms after stimulus onset followed from differences in the AEP topography to each stimulus position, indicative of changes in the underlying brain network. In experiment 2, mirror-symmetric locations were used for training and AEP sessions; no training-related AEP modulations or MMN were observed. In experiment 3, the discrimination of trained plus equidistant untrained separations was tested psychophysically before and 0, 6, 24, and 48 h after training. Learning-induced plasticity lasted <6 h, did not generalize to untrained lateralizations, and was not the simple result of strengthening the representation of the trained lateralizations. Thus, learning-induced plasticity of auditory spatial discrimination relies on spatial comparisons, rather than a spatial anchor or a general comparator. Furthermore, cortical auditory representations of space are dynamic and subject to rapid reorganization.

Transport of a biocontrol Pseudomonas fluorescens through 2.5-m deep outdoor lysimeters and survival in the effluent water

Application of wild-type or genetically-modified bacteria to the soil environment entails the risk of dissemination of these organisms to the groundwater. To measure vertical transport of bacteria under natural climatic conditions, Pseudomonas fluorescens strain CHA0 was released together with bromide as a mobile tracer at the surface of large outdoor lysimeters. Two experiments, one starting in autumn 1993 and the other in spring 1994 were performed. Shortly after a heavy rainfall in late spring 1994, the released bacteria were detected for the first time in effluent water from the 2.5-m-deep lysimeters in both experiments, i.e. 210 d and 21 d, respectively, after inoculation. Only a 10−9 to 10−8 fraction of the inoculum was recovered as culturable cells in the effluent water, but a larger fraction of the CHA0 cells was in a non-culturable state as detected with immunofluorescence microscopy. As much as 50% of the mobile tracer percolated through the lysimeters, indicating that, compared with bromide, bacterial cells were retained in soil. In the second part of this study, persistence of CHA0 in groundwater microcosms consisting of lysimeter effluent water was studied for 380 d. Survival of the inoculant as culturable cells was better under anaerobic than under aerobic conditions. However, a large fraction of the cells became non-culturable in both cases. When the experiment was performed with filter-sterilized effluent water, the total count of introduced bacteria did not decline with time. In conclusion, the biocontrol strain was transported in low numbers to a potential groundwater level under natural climatic conditions, but could persist for an extended period in groundwater microcosms.

Old world arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport.

The highly pathogenic Old World arenavirus Lassa virus (LASV) and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) use α-dystroglycan as a cellular receptor and enter the host cell by an unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the viruses are delivered to acidified endosomes in a Rab5-independent manner bypassing classical routes of incoming vesicular trafficking. Here we sought to identify cellular factors involved in the unusual and largely unknown entry pathway of LASV and LCMV. Cell entry of LASV and LCMV required microtubular transport to late endosomes, consistent with the low fusion pH of the viral envelope glycoproteins. Productive infection with recombinant LCMV expressing LASV envelope glycoprotein (rLCMV-LASVGP) and LCMV depended on phosphatidyl inositol 3-kinase (PI3K) as well as lysobisphosphatidic acid (LBPA), an unusual phospholipid that is involved in the formation of intraluminal vesicles (ILV) of the multivesicular body (MVB) of the late endosome. We provide evidence for a role of the endosomal sorting complex required for transport (ESCRT) in LASV and LCMV cell entry, in particular the ESCRT components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Productive infection with rLCMV-LASVGP and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in membrane dynamics of the MVB/late endosomes. Our study identifies crucial cellular factors implicated in Old World arenavirus cell entry and indicates that LASV and LCMV invade the host cell passing via the MVB/late endosome. Our data further suggest that the virus-receptor complexes undergo sorting into ILV of the MVB mediated by the ESCRT, possibly using a pathway that may be linked to the cellular trafficking and degradation of the cellular receptor.

Evaluation of uptake and transport of cationic and anionic ultrasmall iron oxide nanoparticles by human colon cells.

Nanoparticles (NPs) are in clinical use or under development for therapeutic imaging and drug delivery. However, relatively little information exists concerning the uptake and transport of NPs across human colon cell layers, or their potential to invade three-dimensional models of human colon cells that better mimic the tissue structures of normal and tumoral colon. In order to gain such information, the interactions of biocompatible ultrasmall superparamagnetic iron oxide nanoparticles (USPIO NPs) (iron oxide core 9-10 nm) coated with either cationic polyvinylamine (aminoPVA) or anionic oleic acid with human HT-29 and Caco-2 colon cells was determined. The uptake of the cationic USPIO NPs was much higher than the uptake of the anionic USPIO NPs. The intracellular localization of aminoPVA USPIO NPs was confirmed in HT-29 cells by transmission electron microscopy that detected the iron oxide core. AminoPVA USPIO NPs invaded three-dimensional spheroids of both HT-29 and Caco-2 cells, whereas oleic acid-coated USPIO NPs could only invade Caco-2 spheroids. Neither cationic aminoPVA USPIO NPs nor anionic oleic acid-coated USPIO NPs were transported at detectable levels across the tight CacoReady? intestinal barrier model or the more permeable mucus-secreting CacoGoblet? model.

Transport of the organic cation N1-methylnicotinamide by the rabbit proximal tubule. I. Accumulation in the isolated nonperfused tubule.

Isolated nonperfused rabbit renal proximal tubules were used to investigate the basolateral step of transport of the organic cation N1-methylnicotinamide (NMN). NMN accumulation was highest and saturable in S2 and S3 segments, but lowest and nonsaturable in S1 segments. In S1 segments, accumulation of [3H]-NMN (0.5-8 microM in the bath) resulted in an average tubular water/medium concentration ratio (T/M) of 8.2, whereas in S2 and S3 segments T/M averaged 19.5 and 18.6, respectively. At these concentrations, about 30% of the label was attached in all segments to a metabolite comigrating with nicotinamide. KCN (10(-2) M) or ouabain (10(-4) M) reduced T/M to about 8 for all segments. NMN accumulation was inhibited (to a T/M of about 3 with mepiperphenidol) by other organic cations (10(-5)-10(-3) M) with the potency sequence mepiperphenidol greater than tetraethylammonium = quinine greater than morphine, these organic cations having no effect on p-aminohippurate accumulation, except for the highest concentration of quinine (10(-3) M). After correction for metabolism, NMN accumulation could be accounted for by simple electrochemical equilibrium across the basolateral membrane. The basolateral step of NMN transport appears therefore to be a carrier-mediated diffusion, in opposition to the active basolateral accumulation described for tetraethylammonium.

Role of the ubiquitin system in regulating ion transport.

Ion channels and transporters play a critical role in ion and fluid homeostasis and thus in normal animal physiology and pathology. Tight regulation of these transmembrane proteins is therefore essential. In recent years, many studies have focused their attention on the role of the ubiquitin system in regulating ion channels and transporters, initialed by the discoveries of the role of this system in processing of Cystic Fibrosis Transmembrane Regulator (CFTR), and in regulating endocytosis of the epithelial Na(+) channel (ENaC) by the Nedd4 family of ubiquitin ligases (mainly Nedd4-2). In this review, we discuss the role of the ubiquitin system in ER Associated Degradation (ERAD) of ion channels, and in the regulation of endocytosis and lysosomal sorting of ion channels and transporters, focusing primarily in mammalian cells. We also briefly discuss the role of ubiquitin like molecules (such as SUMO) in such regulation, for which much less is known so far.

Shaping fission yeast cells by rerouting actin-based transport on microtubules.

Kinesins and myosins transport cargos to specific locations along microtubules and actin filaments, respectively. The relative contribution of the two transport systems for cell polarization varies extensively in different cell types, with some cells relying exclusively on actin-based transport while others mainly use microtubules. Using fission yeast, we asked whether one transport system can substitute for the other. In this organism, microtubules and actin cables both contribute to polarized growth by transporting cargos to cell poles, but with distinct roles: microtubules transport landmarks to label cell poles for growth and actin assembly but do not directly contribute to the growth process [1]. Actin cables serve as tracks for myosin V delivery of growth vesicles to cell poles [2-4]. We engineered a chimera between the motor domain of the kinesin 7 Tea2 and the globular tail of the myosin V Myo52, which we show transports Ypt3, a myosin cargo receptor, to cell poles along microtubules. Remarkably, this chimera restores polarized growth and viability to cells lacking actin cables. It also bypasses the normal microtubule-dependent marking of cell poles for polarized growth, but not for other functions. Thus, a synthetic motor protein successfully redirects cargos along a distinct cytoskeletal route.