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Surface Enhanced Raman Scattering (SERS) has been widely praised for its extreme sensitivity but has not so far been put to use in routine analytical applications, with the accessible scale of measurements a limiting factor. We report here on a frugal implementation of SERS dedicated to the quantitative detection of Zn 2+ in water, Zn being an element that can serve as an indicator of contamination by heavy metals in aquatic bodies. The method consists in randomly aggregating simple silver colloids in the analyte solution in the presence of a complexometric indicator of Zn 2+ , recording the SERS spectrum with a portable Raman spectrometer and analysing the data using multivariate calibration models. The frugality of the sensing procedure enables us to acquire a dataset much larger than conventionally done in the field of SERS, which in turn allows for an in-depth statistical analysis of the analytical performances that matter to end-users. In pure water, the proposed sensor is sensitive and accurate in the 160-2230 nM range, with a trueness of 96% and a precision of 4%. Although its limit of detection is one order of magnitude higher than those of golden standard techniques for quantifying metals, its sensitivity range matches Zn levels that are relevant to the health of aquatic bodies. Moreover, its frugality positions it as an interesting alternative to monitor water quality. Critically, the combination of the simple procedure for sample preparation, abundant SERS material and affordable portable instrument paves the way for a realistic deployment to the water site, with each Zn reading three to five times cheaper than through conventional techniques. It could therefore complement current monitoring methods in a bid to solve the pressing needs for large scale water quality data.

Background: The sense of smell is unrivaled in terms of molecular complexity of its input channels. Even zebrafish, a model vertebrate system in many research fields including olfaction, possesses several hundred different olfactory receptor genes, organized in four different gene families. For one of these families, the initially discovered odorant receptors proper, segregation of expression into distinct spatial subdomains within a common sensory surface has been observed both in teleost fish and in mammals. However, for the remaining three families, little to nothing was known about their spatial coding logic. Here we wished to investigate, whether the principle of spatial segregation observed for odorant receptors extends to another olfactory receptor family, the V2R-related OlfC genes. Furthermore we thought to examine, how expression of OlfC genes is integrated into expression zones of odorant receptor genes, which in fish share a single sensory surface with OlfC genes., Results: To select representative genes, we performed a comprehensive phylogenetic study of the zebrafish OlfC family, which identified a novel OlfC gene, reduced the number of pseudogenes to 1, and brought the total family size to 60 intact OlfC receptors. We analyzed the spatial pattern of OlfC-expressing cells for seven representative receptors in three dimensions (height within the epithelial layer, horizontal distance from the center of the olfactory organ, and height within the olfactory organ). We report non-random distributions of labeled neurons for all OlfC genes analysed. Distributions for sparsely expressed OlfC genes are significantly different from each other in nearly all cases, broad overlap notwithstanding. For two of the three coordinates analyzed, OlfC expression zones are intercalated with those of odorant receptor zones, whereas in the third dimension some segregation is observed., Conclusion: Our results show that V2R-related OlfC genes follow the same spatial logic of expression as odorant receptors and their expression zones intermingle with those of odorant receptor genes. Thus, distinctly different expression zones for individual receptor genes constitute a general feature shared by teleost and tetrapod V2R/OlfC and odorant receptor families alike.

Biomineralization is the process of mineral formation by organisms and involves the uptake of ions from the environment in order to produce minerals, with the process generally being mediated by proteins. Most proteins that are involved in mineral interactions are predicted to contain disordered regions containing large numbers of negatively charged amino acids. Magnetotactic bacteria, which are used as a model system for iron biomineralization, are Gram-negative bacteria that can navigate through geomagnetic fields using a specific organelle, the magnetosome. Each organelle comprises a membrane-enveloped magnetic nanoparticle, magnetite, the formation of which is controlled by a specific set of proteins. One of the most abundant of these proteins is MamC, a small magnetosome-associated integral membrane protein that contains two transmembrane α-helices connected by an ∼21-amino-acid peptide. In vitro studies of this MamC peptide showed that it forms a helical structure that can interact with the magnetite surface and affect the size and shape of the growing crystal. Our results show that a disordered structure of the MamC magnetite-interacting component (MamC-MIC) abolishes its interaction with magnetite particles. Moreover, the size and shape of magnetite crystals grown in in vitro magnetite-precipitation experiments in the presence of this disordered peptide were different from the traits of crystals grown in the presence of other peptides or in the presence of the helical MIC. It is suggested that the helical structure of the MamC-MIC is important for its function during magnetite formation.

Magnetite nanoparticles exhibit magnetic properties that are size and organization dependent and, for applications that rely on their magnetic state, they usually have to be monodisperse. Forming such particles, however, has remained a challenge. Here, we synthesize 40 nm particles of magnetite in the presence of polyarginine and show that they are composed of 10 nm building blocks, yet diffract like single crystals. We use both bulk magnetic measurements and magnetic induction maps recorded from individual particles using off-axis electron holography to show that each 40 nm particle typically contains a single magnetic domain. The magnetic state is therefore determined primarily by the size of the superstructure and not by the sizes of the constituent sub-units. Our results fundamentally demonstrate the structure - property relationship in a magnetic mesoparticle.

It is now recognized that nucleation and growth of crystals can occur not only by the addition of solvated ions but also by accretion of nanoparticles, in a process called nonclassical crystallization. The theoretical framework of such processes has only started to be described, partly due to the lack of kinetic or thermodynamic data. Here, we study the growth of magnetite nanoparticles from primary particles-nanometer-sized amorphous iron-rich precursors-in aqueous solution at different temperatures. We propose a theoretical framework to describe the growth of the nanoparticles and model both a diffusion-limited and a reaction-limited pathway to determine which of these best describes the rate-limiting step of the process. We show that, based on the measured iron concentration and the related calculated concentration of primary particles at the steady state, magnetite growth is likely a reaction-limited process, and within the framework of our model, we propose a phase diagram to summarize the observations.

Background: Opioid maintenance treatment with methadone is regarded as gold standard in the therapy of opioid dependence. Identification of the 'right' methadone dose, however, remains challenging. We wanted to explore if the Opiate Dosage Adequacy Scale (ODAS) is a helpful instrument in methadone titration., Methods: Within this 12-months prospective naturalistic cohort study patients in stable maintenance treatment with methadone (Eptadone®) were included. Sociodemographic and clinical data were gathered at baseline, and months 3, 6, and 12. At the same points in time, the instruments ODAS, European Addiction Severity Index (EuropASI), and Derogatis Interview for Sexual Functioning-Self Report (DISF-SR) were applied., Results: Five hundred fifteen patients were enrolled, 129 patients prematurely terminated substitution treatment (treatment failure), in 108 patients substitution medication was changed, likely due to bitter taste of Eptadone®. Complete longitudinal ODAS and EuropASI data sets were available for 229 patients. The frequency of adequate methadone doses (ODAS) increased (60.9 % at baseline, 85.3 % at month 12) as well as the average daily methadone dose (63.8 (±30.8) mg/day at baseline to 69.6 (±36.0) mg/day at month 12). Inadequacy of methadone dose was not associated with treatment failure (RR 1.019; CI 95 % 0.756-1.374). Addiction severity decreased statistically significantly. Compared to adequately dosed patients, inadequately dosed patients benefited more, in that they showed greater improvements in ODAS scores, had higher increases in methadone dose, and partially experienced more advanced sexual functioning., Conclusion: Application of ODAS was associated with improved methadone dose adequacy and addiction severity parameters as well as increased methadone doses. Its usefulness should be corroborated in a controlled trial.

Access to spiritual support appears to be important in the hospital setting. The offer of spiritual support can be done by different providers such as doctors, nurses or chaplains. Who should initiate or coordinate this spiritual care. This study addresses the following questions: 1) How many patients accept spiritual proposition? 2) What is the better mode of proposition? The study's objectives are the assessment and comparison of the rates of acceptance to an offer of spiritual support made by nurses and chaplains. Two hundred twenty-three consecutive hospitalized patients hospitalized received a proposal of spiritual support and were randomly assigned to one of two conditions. Results revealed that 85.8% of patients accepted the offer in the chaplains' group and 38.5% in the nurses' group. Acceptance of the offer of spiritual support was positively associated with the proposal being made by the chaplains by the frequency of meditation and age, and negatively related to physical well-being.

Traumatic mating behaviors often bear signatures of sexual conflict and are then typically considered a male strategy to circumvent female choice mechanisms. In an extravagant mating ritual, the hermaphroditic sea slug Siphopteron quadrispinosum pierces the integument of their mating partners with a syringe-like penile stylet that injects prostate fluids. Traumatic injection is followed by the insertion of a spiny penis into the partner's gonopore to transfer sperm. Despite traumatic mating, field mating rates exceed those required for female fertilization insurance, possibly because costs imposed on females are balanced by direct or indirect benefits of multiple sperm receipt. To test this idea, we exposed animals to a relevant range of mating opportunity regimes and assessed the effects on mating behavior and proxies of female fitness. We find penis intromission duration to decrease with mating rates, and a female fecundity maximum at intermediate mating rates. The latter finding indicates that benefits beyond fertilization insurance can make higher mating rates also beneficial from a female perspective in this traumatically mating species.

The aim of the study was to investigate the influence of diabetes mellitus type 1 on expression and function of the ATP-binding cassette transport proteins P-glycoprotein (Pgp, Abcb1) and breast cancer resistance protein (Bcrp, Abcg2) at the blood-brain barrier and the blood-cerebrospinal fluid barrier formed by the choroid plexus. In brain capillary endothelial cells forming the blood-brain barrier, Pgp and Bcrp are located in the luminal membrane while apical/sub-apical localization was described for Pgp in choroid plexus epithelial cells. Alterations in expression or function may lead to damages in barrier integrity and may cause brain defects after long term diabetes. Diabetes was induced by i.p.-streptozotocin injection 14days prior to performing experiments. RNA and protein expression were analyzed in choroid plexus and blood-brain barrier capillaries by RT-PCR and Western blot, respectively. Pgp and Bcrp expression was increased in blood-brain barrier capillaries; in choroid plexus, only Bcrp showed elevated gene expression. Protein expression was not altered. Functional analyses were carried out using confocal laser-scanning microscopy in intact isolated brain capillaries with the fluorescent Pgp substrate NBD-Cyclosporin A (NBD-CsA) and BODIPY® FL prazosin as substrate for Bcrp. Consistent with protein expression data, no changes in diabetic animals occurred, suggesting an unaltered function of Pgp and Bcrp., (Copyright © 2010 Elsevier B.V. All rights reserved.)

According to the World Health Organization Central nervous system disorders are the major medical challenge of the 21st Century, yet treatments for many CNS disorders are either inadequate or absent. One reason is the existence of the blood-brain barrier, which strictly limits the access of substances to the brain. A key element of the barrier function is the expression of ABC export proteins in the luminal membrane of brain microvessel endothelial cells. Understanding the signaling cascades and the response to endogenous and exogenous stimuli, which lead to altered expression or function of the transporters as well as subsequent modulation of the transporters, may offer novel strategies to overcome the barrier and to improve drug delivery to the brain. This review gives a short overview about structure of the key elements of the blood-brain barrier with emphasis on ABC transporters. An insight into regulation of function and expression of these transport proteins is given and the involvement of these transporters in CNS diseases is discussed.

The choroid plexuses (CP) are responsible for transport of micronutrients into brain and clearance of toxic compounds, in addition to its barrier function and production of CSF. Multidrug resistance-associated protein (Mrp) 4 is one transport protein highly expressed in CP tissue and is characterized as a versatile pump for toxicants and signalling molecules. Aim of the study was to determine transport characteristics of a fluorescent cAMP analog in rat CP and to define whether fluo-cAMP can be used for analyses of function, substrate/inhibitor specificity and regulation of Mrp4. Confocal imaging was used to analyze transport mechanisms in absence and presence of various modulators of organic anion transport in freshly isolated and functionally intact CP. Fluo-cAMP transport was saturable, selective, concentrative and metabolism-dependent, following an active two-step mechanism composed of apical uptake into epithelial cells and basolateral efflux. Uptake included a Na(+) -dependent and a Na(+) -independent component and was inhibited by estrone sulfate, taurocholate and sildenafil indicating involvement of organic anion transporting polypeptide Oatp1a5. Efflux was composed of an indirect Na(+) -dependent component and a component inhibitable by, for example, the MRP4 substrates/inhibitors, sulindac sulfide and 4-(2-aminoethyl) benzenesulfonyl fluoride. Therefore, fluo-cAMP can be used as fluorescent model compound for studying involvement of Mrp4 in signalling pathways and neuroprotection in CP., (© 2010 The Authors. Journal Compilation © 2010 International Society for Neurochemistry.)

Background: Two rodent choroid plexus (CP) epithelial cell lines, Z310 and TR-CSFB, were compared with primary rat CP epithelial cells and intact CP tissue with respect to transport protein expression, function and tight junction (TJ) formation., Methods: For expression profiles of transporters and TJ proteins, qPCR and western blot analysis were used. Uptake assays were performed to study the functional activity of transporters and TJ formation was measured by trans-epithelial electrical resistance (TEER) and visualized by electron microscopy., Results: The expression of known ATP-binding cassette (Abc) transporter and solute carrier (Slc) genes in CP was confirmed by qPCR. Primary cells and cell lines showed similar, but overall lower expression of Abc transporters and absent Slc expression when compared to intact tissue. Consistent with this Mrp1, Mrp4 and P-gp protein levels were higher in intact CP compared to cell lines. Functionality of P-gp and Mrp1 was confirmed by Calcein-AM and CMFDA uptake assays and studies using [3H]bis-POM-PMEA as a substrate indicated Mrp4 function. Cell lines showed low or absent TJ protein expression. After treatment of cell lines with corticosteroids, RNA expression of claudin1, 2 and 11 and occludin was elevated, as well as claudin1 and occludin protein expression. TJ formation was further investigated by freeze-fracture electron microscopy and only rarely observed. Increases in TJ particles with steroid treatment were not accompanied by an increase in transepithelial electrical resistance (TEER)., Conclusion: Taken together, immortalized cell lines may be a tool to study transport processes mediated by P-gp, Mrp1 or Mrp4, but overall expression of transport proteins and TJ formation do not reflect the situation in intact CP tissue.

Purpose: To establish a fluorescence-based assay for drug interactions with the ABC-export-protein MRP2 (ABCC2)., Methods: Apical membrane vesicles were isolated by differential centrifugation from polarized MDCKII cells and MDCKII cells transfected with human MRP2. Vesicle fractions were characterized by electron microscopy, determination of the marker enzyme alkaline phosphatase and Western blot analysis of MRP2. Vesicle orientation was determined by measurement of 5'-nucleotidase activity in the absence and in the presence of detergents. To assess MRP2 activity, the uptake of the fluorescent MRP2-substrate 5-(6)-carboxy-2',7'-dichlorofluorescein (CDF) was determined in the absence and in the presence of other compounds potentially interacting with MRP2., Results: Apical membrane vesicles could be isolated from cells in considerable purity as indicated by electron microscopy, enrichment of alkaline phosphatase and high enrichment of MRP2 in vesicles of MDCKII-MRP2 cells. About half of the vesicles showed "inside-out" orientation. CDF was taken up into the membrane vesicles in a time- and concentration-dependent manner following a Michaelis-Menten type of kinetics with a K(M) of 39 microM and a V(max) of 465.3 fmol/(mgprotein x min). Thereby, uptake into vesicles from transfected cells was significantly higher than uptake into vesicles from control cells. Presence of known MRP2-substrates/inhibitors in the incubation medium decreased CDF uptake into the vesicles in a concentration-dependent manner, whereas nonsubstrates/inhibitors had no effect., Conclusions: This CDF-based uptake assay can be used as a rapid and sensitive screening system to assess drug interactions with human MRP2 and therefore represents a useful tool in compound profiling., (Copyright 2010 Elsevier B.V. All rights reserved.)

We report about a new type of nonlinear photonic crystal fibers allowing broadband four-wave mixing and supercontinuum generation. The microstructured optical fiber has a structured core consisting of a rod of highly nonlinear glass material inserted in a silica tube. This particular structure enables four wave mixing processes with very large frequency detuning (>135 THz), which permitted the generation of a wide supercontinuum spectrum extending over 1650 nm after 2.15 m of propagation length. The comparison with results obtained from germanium-doped holey fibers confirms the important role of the rod material properties regarding nonlinear process and dispersion.

We report on the thermal treatment of photodarkened Yb-doped fiber samples. The method of non-isothermal bleaching at different temperature ramp rates can be used to determine the thermal energy distribution of photodarkening induced color centers. A distributed activation energy with a mean value of about 1.3 eV and a FWHM of 0.5 eV was found. Spectral changes during thermal treatment were observed and could be interpreted, e.g. as an enhancement of the absorption cross section.

Confocal microscopy and image analysis were used to compare driving forces, specificity, and regulation of transport of the fluorescent organic anion, Texas Red (sulforhodamine 101 free acid; TR), in lateral choroid plexus (CP) isolated from rat and an evolutionarily ancient vertebrate, dogfish shark (Squalus acanthias). CP from both species exhibited concentrative, specific, and metabolism-dependent TR transport from bath to subepithelial/vascular space; at steady state, TR accumulation in vascular/subepithelial space was substantially higher than in epithelial cells. In rat CP, steady-state TR accumulation in subepithelial/vascular spaces was reduced by Na(+)-replacement, but was not affected by a 10-fold increase in buffer K(+). In shark CP, Na(+)-replacement did not alter TR accumulation in either tissue compartment; subepithelial/vascular space levels of TR were reduced in high-K(+) medium. In both species, steady-state TR accumulation was not affected by p-aminohippurate or leukotriene C4, suggesting that neither organic anion transporters (SLC22A family) nor multidrug resistance-associated proteins (ABCC family) contributed. In rat CP, digoxin was without effect, indicating that organic anion transporting polypeptide isoform 2 was not involved. Several organic anions reduced cellular and subepithelial/vascular space TR accumulation in both tissues, including estrone sulfate, taurocholate, and the Mrp1 inhibitor MK571. In rat CP, TR accumulation in subepithelial/vascular spaces increased with PKA activation (forskolin), but was not affected by PKC activation (phorbol ester). In shark, neither PKA nor PKC activation specifically affected TR transport. Thus, rat and dogfish shark CP transport TR but do so using different basic mechanisms that respond to different regulatory signals.

Previous studies have shown that killifish (Fundulus heteroclitus) renal proximal tubules express a luminal membrane transporter that is functionally and immunologically analogous to the mammalian multidrug resistance-associated protein isoform 2 (Mrp2, ABCC2). Here we used confocal microscopy to investigate in killifish tubules the transport of a fluorescent cAMP analog (fluo-cAMP), a putative substrate for Mrp2 and Mrp4 (ABCC4). Steady-state luminal accumulation of fluo-cAMP was concentrative, specific, and metabolism-dependent, but not reduced by high K+ medium or ouabain. Transport was not affected by p-aminohippurate (organic anion transporter inhibitor) or p-glycoprotein inhibitor (PSC833), but cell-to-lumen transport was reduced in a concentration-dependent manner by Mrp inhibitor MK571, leukotriene C4 (LTC4), azidothymidine (AZT), cAMP, and adefovir; the latter two compounds are Mrp4 substrates. Although MK571 and LTC4 reduced transport of the Mrp2 substrate fluorescein-methotrexate (FL-MTX), neither cAMP, adefovir, nor AZT affected FL-MTX transport. Fluo-cAMP transport was not reduced when tubules were exposed to endothelin-1, Na nitroprusside (an nitric oxide generator) or phorbol ester (PKC activator), all of which signal substantial reductions in cell-to-lumen FL-MTX transport. Fluo-cAMP transport was reduced by forskolin, and this reduction was blocked by the PKA inhibitor H-89. Finally, in membrane vesicles from Spodoptera frugiperda (Sf9) cells containing human MRP4, ATP-dependent and specific uptake of fluo-cAMP could be demonstrated. Thus, based on inhibitor specificity and regulatory signaling, cell-to-lumen transport of fluo-cAMP in killifish renal tubules is mediated by a transporter distinct from Mrp2, presumably a teleost form of Mrp4.

The choroid plexus represents the second barrier between blood and brain besides the blood brain barrier. The barrier function is set up by plexus epithelial cells, which are equipped with a variety of active transport systems. However in contrast to the epithelial organs location and extent of expression of these transporters may be different. Studying the choroid plexus (CP) epithelium in living animals is difficult due to its complex morphology, anatomical location and small size. Therefore, an in vitro monolayer model of choroid plexus was established from porcine brain in order to study the functional contribution of transport proteins to drug transport across CP tissue with special focus on ABC-proteins.

Background: The goal of the present study was to develop an in vitro choroid plexus (CP) epithelial cell culture model for studying transport of protein-mediated drug secretion from blood to cerebrospinal fluid (CSF) and vice versa., Methods: Cells were isolated by mechanical and enzymatic treatment of freshly isolated porcine plexus tissue. Epithelial cell monolayers were grown and CSF secretion and transepithelial resistance were determined. The expression of f-actin as well as the choroid plexus marker protein transthyretin (TTR), were assessed. The expression of the export proteins p-glycoprotein (Pgp, Abcb1) and multidrug resistance protein 1 (Mrp1, Abcc1) was studied by RT-PCR, Western-blot and immunofluorescence techniques and their functional activity was assessed by transport and uptake experiments., Results: Choroid plexus epithelial cells were isolated in high purity and grown to form confluent monolayers. Filter-grown monolayers displayed transendothelial resistance (TEER) values in the range of 100 to 150 ohms cm2. Morphologically, the cells showed the typical net work of f-actin and expressed TTR at a high rate. The cultured cells were able to secrete CSF at a rate of 48.2 +/- 4.6 microl/cm2/h over 2-3 hours. The ABC-export protein Mrp1 was expressed in the basolateral (blood-facing) membranes of cell monolayers and intact tissue. P-glycoprotein showed only low expression within the apical (CSF directed) membrane but was located more in sub-apical cell compartments. This finding was paralleled by the lack of directed excretion of p-glycoprotein substrates, verapamil and rhodamine 123., Conclusion: It was demonstrated that CP epithelium can be isolated and cultured, with cells growing into intact monolayers, fully differentiating and with properties resembling the tissue in vivo. Thus, the established primary porcine CP model, allowing investigation of complex transport processes, can be used as a reliable tool for analysis of xenobiotic transport across the blood-cerebrospinal fluid barrier (BCSFB).

Comparative ventricular measurements were made on 43 small children and infants between the ages of 0 and 3 years, the methods used being those of pneumoencephalography and enchoventriculography. The number of cases that could be analyzed was 31. Good agreement of the results for the third ventricle was obtained in 64.5 per cent and for the lateral ventricular index, in 80.6 per cent of the cases. More or less satisfactory agreement of the two methods provides against making a clear decision in favor of one or the other method. Good agreement was observed between the results of repeated echoventriculographic examinations on 31 normal children. From these results as well as from th results of echographic examination on hydrocephalic children it can be concluded that echoventriculography is a reliable method of diagnosis even in those cases in which it is routinely applied to both inpatients and outpatients.