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Introduction: Astrocytic GLT-1 glutamate transporters ensure the fidelity of glutamic neurotransmission by spatially and temporally limiting glutamate signals. The ability to limit neuronal hyperactivity relies on the localization and diffusion of GLT-1 on the astrocytic surface, however, little is known about the underlying mechanisms. We show that two isoforms of GLT-1, GLT-1a and GLT- 1b, form nanoclusters on the surface of transfected astrocytes and HEK-293 cells. Methods: We used both fixed and live cell super-resolution imaging of fluorescent protein and epitope tagged proteins in co-cultures of rat astrocytes and neurons. Immunofluorescence techniques were also used. GLT1 diffusion was assessed via single particle tracking and fluorescence recovery after photobleach (FRAP). Results: We found GLT-1a, but not GLT-1b, nanoclusters concentrated adjacent to actin filaments which was maintained after addition of glutamate. GLT-1a nanocluster concentration near actin filaments was prevented by expression of a cytosolic GLT-1a C-terminus, suggesting the C-terminus is involved in the localization adjacent to cortical actin. Using super-resolution imaging, we show that astrocytic GLT-1a and actin co-localize in net-like structures around neuronal Kv2.1 clusters at points of neuron/astrocyte contact. Conclusion: Overall, these data describe a novel relationship between GLT-1a and cortical actin filaments, which localizes GLT-1a near neuronal structures responsive to ischemic insult. [ABSTRACT FROM AUTHOR]

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Introduction: Astrocytic GLT-1 glutamate transporters ensure the fidelity of glutamic neurotransmission by spatially and temporally limiting glutamate signals. The ability to limit neuronal hyperactivity relies on the localization and diffusion of GLT-1 on the astrocytic surface, however, little is known about the underlying mechanisms. We show that two isoforms of GLT-1, GLT-1a and GLT-1b, form nanoclusters on the surface of transfected astrocytes and HEK-293 cells.Methods: We used both fixed and live cell super-resolution imaging of fluorescent protein and epitope tagged proteins in co-cultures of rat astrocytes and neurons. Immunofluorescence techniques were also used. GLT1 diffusion was assessed via single particle tracking and fluorescence recovery after photobleach (FRAP).Results: We found GLT-1a, but not GLT-1b, nanoclusters concentrated adjacent to actin filaments which was maintained after addition of glutamate. GLT-1a nanocluster concentration near actin filaments was prevented by expression of a cytosolic GLT-1a C-terminus, suggesting the C-terminus is involved in the localization adjacent to cortical actin. Using super-resolution imaging, we show that astrocytic GLT-1a and actin co-localize in net-like structures around neuronal Kv2.1 clusters at points of neuron/astrocyte contact.Conclusion: Overall, these data describe a novel relationship between GLT-1a and cortical actin filaments, which localizes GLT-1a near neuronal structures responsive to ischemic insult.

Nuclear lamins and transport are intrinsically linked, but their relationship is yet to be fully unraveled. A multitude of complex, coupled interactions between lamins and nucleoporins (Nups), which mediate active transport into and out of the nucleus, combined with well documented dysregulation of lamins in many cancers, suggests that lamins and nuclear transport may play a pivotal role in carcinogenesis and the preservation of cancer. Changes of function related to lamin/Nup activity can principally lead to DNA damage, further increasing the genetic diversity within a tumor, which could lead to the reduction the effectiveness of antineoplastic treatments. This review discusses and synthesizes different connections of lamins to nuclear transport and offers a number of outlook questions, the answers to which could reveal a new perspective on the connection of lamins to molecular transport of cancer therapeutics, in addition to their established role in nuclear mechanics. [ABSTRACT FROM AUTHOR]

There is an unmet need for novel therapeutic tools relieving chronic pain. Hydrogen sulfide (H2S) is highly involved in pain processes; however, the development of ideal matrices for sulfide donor compounds remains a great pharmaceutical challenge. We aimed to establish a suitable transdermal therapeutic system (TTS) using the H2S donor diallyl disulfide (DADS) as a model compound. After the preparation of DADS, its solubility was investigated in different liquid excipients (propylene glycol, polyethylene glycol, silicone oil) and its membrane diffusivity was assessed in silicone matrices of different compositions. Drug-releasing properties of DADS-containing patches with different silicone oil contents were determined with Franz and flow-through cells. We found a correlation between the liquid excipient content of the patch and the diffusion rate of DADS. DADS showed the best solubility in dimethyl silicone oil, and the diffusion constant was proportional to the amount of oil above the 3 m/m% threshold value. The 8-day-old patch showed a significantly lower, but better-regulated, drug release over time than the 4-day-old one. In conclusion, the silicone-based polymer matrix developed in this study is suitable for stable storage and optimal release of DADS, providing a good basis for a TTS applied in chronic pain. [ABSTRACT FROM AUTHOR]

Neuroligins (NLGNs) form a family of cell adhesion molecules implicated in synapse development, but the mechanisms that retain these proteins at synapses are still incompletely understood. Recent studies indicate that surface-associated NLGN1 is diffusionally trapped at synapses, where it interacts with quasi-static scaffolding elements of the post-synaptic density. Whereas single molecule tracking reveals rapid diffusion and transient immobilization of NLGN1 at synapses within seconds, fluorescence recovery after photobleaching experiments indicate instead a long-term turnover of NLGN1 at synapse, in the hour time range. To gain insight into the mechanisms supporting NLGN1 anchorage at post-synapses and try to reconcile those experimental paradigms, we quantitatively analyzed here live-cell and super-resolution imaging experiments performed on NLGN1 using a newly released simulator of membrane protein dynamics for fluorescence microscopy, FluoSim. Based on a small set of parameters including diffusion coefficients, binding constants, and photophysical rates, the framework describes fairly well the dynamic behavior of extra-synaptic and synaptic NLGN1 over both short and long time ranges, and provides an estimate of NLGN1 copy numbers in post-synaptic densities at steady-state (around 50 dimers). One striking result is that the residence time of NLGN1 at synapses is much longer than what can be expected from extracellular interactions with pre-synaptic neurexins only, suggesting that NLGN1 is stabilized at synapses through multivalent interactions with intracellular post-synaptic scaffolding proteins.

There is an unmet need for novel therapeutic tools relieving chronic pain. Hydrogen sulfide (H2S) is highly involved in pain processes; however, the development of ideal matrices for sulfide donor compounds remains a great pharmaceutical challenge. We aimed to establish a suitable transdermal therapeutic system (TTS) using the H2S donor diallyl disulfide (DADS) as a model compound. After the preparation of DADS, its solubility was investigated in different liquid excipients (propylene glycol, polyethylene glycol, silicone oil) and its membrane diffusivity was assessed in silicone matrices of different compositions. Drug-releasing properties of DADS-containing patches with different silicone oil contents were determined with Franz and flow-through cells. We found a correlation between the liquid excipient content of the patch and the diffusion rate of DADS. DADS showed the best solubility in dimethyl silicone oil, and the diffusion constant was proportional to the amount of oil above the 3 m/m% threshold value. The 8-day-old patch showed a significantly lower, but better-regulated, drug release over time than the 4-day-old one. In conclusion, the silicone-based polymer matrix developed in this study is suitable for stable storage and optimal release of DADS, providing a good basis for a TTS applied in chronic pain.

Lead (Pb) is one of the most toxic, hazardous pollutants available in landfill leachate. Loess-amended soil buffers are found suitable and effective in attenuating migration of Pb and the other trace metals. High concentration of ammonium (NH4+ > 1000 mg/l) is also reported in landfill leachate, and therefore, it is essential to investigate the transport of lead under such condition. In this study, the mechanisms and the capacity of loess to adsorb Pb under high NH4+ concentration were investigated. Adsorption isotherm test data were obtained for 25 °C, 35 °C and 45 °C. The maximum adsorption capacity is estimated to be 2101.97 mg/g at 25 °C and 4292.8 mg/g at 45 °C under 1000 mg/l NH4+. The binding sites of Pb on loess are positively related to each other at low temperatures (25–35 °C). The thermodynamic analysis indicates that adsorption process is endothermic and non-spontaneous and the system randomness increases with reaction time. The kinetic test data, fitted with a pseudo-second-order kinetic model and an intraparticle diffusion model, suggests that removal of Pb is driven by both membrane and intraparticle diffusions. The SEM, XRD and FTIR analyses indicate flocculation, precipitations as well as some ion exchange processes, which perhaps combinedly increases adsorption of both NH4+ and Pb in loess. The two kinds of precipitations are involved for the removal of Pb. The precipitations of PbCO3, Pb(OH)2 and PbCO3·2H2O are formed by the reactions between calcite and lead. The other precipitation of white basic salt (Pb2O(NO3)2) is formed by the reactions among Pb2+, NO3− and aqueous ammonia under alkaline environment of loess slurry. [ABSTRACT FROM AUTHOR]

The opening and closure of stomata depend on the turgor pressure adjustment by the influx or efflux of ions and water in guard cells. In this process, aquaporins may play important roles by facilitating the transport of water and other small molecules. In this perspective, we consider the potential roles of aquaporins in the membrane diffusion of different molecules (H2O, CO2, and H2O2), processes dependent on abscisic acid and CO2 signaling in guard cells. While the limited data already available emphasizes the roles of aquaporins in stomatal movement, we propose additional approaches to elucidate the specific roles of single or several aquaporin isoforms in the stomata and evaluate the perspectives aquaporins might offer to improve stomatal dynamics.

Tivadar Bíró,1 Gabriella Horvát,1 Mária Budai-Szűcs,1 Erzsébet Csányi,1 Edit Urbán,2 Andrea Facskó,3 Piroska Szabó-Révész,1 Ildikó Csóka,1 Zoltán Aigner1 1Institute of Pharmaceutical Technology and Regulatory Affairs, University of Szeged, Szeged, Hungary; 2Department of Clinical Microbiology, Faculty of Medicine, University of Szeged, Szeged, Hungary; 3Ophthalmology Department, Faculty of Medicine, University of Szeged, Szeged, Hungary Purpose: The formulation of topical ophthalmic products with appropriate therapeutic effect and patient compliance is a major challenge. To increase the efficiency of the ocular delivery of the drug, the enhancement of water solubility and the contact time of the drug on the surface of the cornea are necessary. In this work, prednisolone (PR)-containing eye drops were formulated with antimicrobial, mucoadhesive biopolymer and PR–cyclodextrin inclusion complex. This approach can be used for the development of innovative ophthalmic formulations. Materials and methods: After adjusting the optimal physiological parameters, the amount of the required cyclodextrin for the highest penetration of PR was determined by dialysis membrane diffusion study. The viscosity, surface tension and mucoadhesion of the eye drops were measured. The microbiological effectiveness of zinc-hyaluronate (ZnHA) was investigated by a standard method of the European Pharmacopoeia. Results: In this case, no significant difference of surface tension was measured in products with different amounts of cyclodextrin. According to the results of the tensile test, ZnHA as a mucoadhesive biopolymer improves the mucoadhesion of ophthalmic products. The antimicrobial stability of formulations preserved by ZnHA meets requirement B of the European Pharmacopoeia. Conclusion: It can be stated that the innovative PR-containing compositions are suitable for producing mucoadhesive, properly preserved aqueous ophthalmic solutions with increased bioavailability attributes. Keywords: pharmaceutical formulation, zinc-hyaluronate, ocular drug delivery, microbiological stability, membrane diffusion

The opening and closure of stomata depend on the turgor pressure adjustment by the influx or efflux of ions and water in guard cells. In this process, aquaporins may play important roles by facilitating the transport of water and other small molecules. In this perspective, we consider the potential roles of aquaporins in the membrane diffusion of different molecules (H2O, CO2, and H2O2), processes dependent on abscisic acid and CO2 signaling in guard cells. While the limited data already available emphasizes the roles of aquaporins in stomatal movement, we propose additional approaches to elucidate the specific roles of single or several aquaporin isoforms in the stomata and evaluate the perspectives aquaporins might offer to improve stomatal dynamics. [ABSTRACT FROM AUTHOR]

Simplified engineering models are presented, characterizing Donnan dialysis separations of a single and multicomponent feed solutions by an overall transport coefficient combining mass transfer resistance and membrane diffusion resistance. Full verification of the predictions of the derived models was obtained from experimental data measured at various Reynolds numbers and feed solution concentrations using a NaCl stripping solution. Permeating target ions from single component feed solutions consisted of H2PO4−, NO3−, and HCO3−. Dual component permeation was from feed solutions of the ion pairs H2PO4−/NO3−, and NO3−/HCO3−. Permeation of all ions except the H2PO4− ion was found to be mass transfer controlled. [ABSTRACT FROM AUTHOR]

Abstract Understanding the diffusion of nanoparticles through permeable membranes in cell mimics paves the way for the construction of more sophisticated synthetic protocells with control over the exchange of nanoparticles or biomacromolecules between different compartments. Nanoparticles postloading by swollen pH switchable polymersomes is investigated and nanoparticles locations at or within polymersome membrane and polymersome lumen are precisely determined. Validation of transmembrane diffusion properties is performed based on nanoparticles of different origin—gold, glycopolymer protein mimics, and the enzymes myoglobin and esterase—with dimensions between 5 and 15 nm. This process is compared with the in situ loading of nanoparticles during polymersome formation and analyzed by advanced multiple‐detector asymmetrical flow field‐flow fractionation (AF4). These experiments are supported by complementary i) release studies of protein mimics from polymersomes, ii) stability and cyclic pH switches test for in polymersome encapsulated myoglobin, and iii) cryogenic transmission electron microscopy studies on nanoparticles loaded polymersomes. Different locations (e.g., membrane and/or lumen) are identified for the uptake of each protein. The protein locations are extracted from the increasing scaling parameters and the decreasing apparent density of enzyme‐containing polymersomes as determined by AF4. Postloading demonstrates to be a valuable tool for the implementation of cell‐like functions in polymersomes.

Shengli brown coal in 150-180 휇m was gasified at 800℃/900℃ in a simulated entrained-flow reactor, 훷80 mmx 3 000 mm. Kinetics of main reactions and flow characteristics of gas/solid were discussed to build a coal mild gasification model. The results show that,firstly,the gas phase flow was divided into 3 zones,including jet zone, recirculation zone and downstream zone. The height of downstream zone was 95% of that of reactor. Secondly, pyrolysis, combustion and gasification could be considered running in order as a result of different reaction rates. Thirdly,the rate equations of oxidation reaction (OR) and steam gasification reaction (SGR) derived were in good agreement with the experimental data. The OR rate was in membrane diffusion controlled regime, and SGR rate was in chemical controlled regime. Moreover,the apparent rate constant of SGR increased obviously after adding O2 to steam. Therefore,the kinetic equation derived was suitable with the experimental conditions. Fourthly,the reactions of H2 ,CO,CH4 combustion,char-CO2/H2 gasification,CO-H2 methanation,and water-gas shift reaction were considered,while the reactions of tar combustion,char-H2O methanation,CO2-H2 methanation were ignored. Fifthly,due to the difference of char microstructure, the effective contact (collision) area between gas and solid in different temperatures and atmospheres is different significantly. Based on those,a numerical model of en-trained-flow reactor was built,and its predicted values were in good agreement with the experimental values, with the error of less than 20% for 92% predicted values and less than 10% for 75% predicted values. [ABSTRACT FROM AUTHOR]

Background Drug permeation through skin, or a synthetic membrane, from locally acting pharmaceutical products can be influenced by the permeation behaviour of pharmaceutical excipients. Objective Terahertz time-domain technology is investigated as a non-invasive method for a direct and accurate measurement of excipients permeation through synthetic membranes or human skin. Methods A series of in-vitro release and skin permeation experiments of liquid excipients (e.g. propylene glycol and polyethylene glycol 400) has been conducted with vertical diffusion cells. The permeation profiles of excipients through different synthetic membranes or skin were obtained using Terahertz pulses providing a direct measurement. Corresponding permeation flux and permeability coefficient values were calculated based on temporal changes of the terahertz pulses. Results The influence of different experimental conditions, such as the polarity of the membrane and the viscosity of the permeant, was assessed in release experiments. Specific transmembrane flux values of those excipients were directly calculated with statistical differences between cases. Finally, an attempt to estimate the skin permeation of propylene glycol with this technique was also achieved. All these permeation results were likely comparable to those obtained by other authors with usual analytical techniques. Conclusion Terahertz time-domain technology is shown to be a suitable technique for an accurate and non-destructive measurement of the permeation of liquid substances through different synthetic membranes or even human skin. [ABSTRACT FROM AUTHOR]

The diffusion process through a non-porous barrier membrane depends on the properties of the drug, vehicle and membrane. The aim of the current study was to investigate whether a series of oily vehicles might have the potential to interact to varying degrees with synthetic membranes and to determine whether any such interaction might affect the permeation of co-formulated permeants: methylparaben (MP); butylparaben (BP) or caffeine (CF). The oils (isopropyl myristate (IPM), isohexadecane (IHD), hexadecane (HD), oleic acid (OA) and liquid paraffin (LP)) and membranes (silicone, high density polyethylene and polyurethane) employed in the study were selected such that they displayed a range of different structural, and physicochemical properties. Diffusion studies showed that many of the vehicles were not inert and did interact with the membranes resulting in a modification of the permeants’ flux when corrected for membrane thickness (e.g. normalized flux of MP increased from 1.25 ± 0.13 μg cm −1 h −1 in LP to 17.94 ± 0.25 μg cm −1 h −1 in IPM). The oils were sorbed differently to membranes (range of weight gain: 2.2 ± 0.2% for polyurethane with LP to 105.6 ± 1.1% for silicone with IHD). Membrane interaction was apparently dependent upon the physicochemical properties including; size, shape, flexibility and the Hansen solubility parameter values of both the membranes and oils. Sorbed oils resulted in modified permeant diffusion through the membranes. No simple correlation was found to exist between the Hansen solubility parameters of the oils or swelling of the membrane and the normalized fluxes of the three compounds investigated. More sophisticated modelling would appear to be required to delineate and quantify the key molecular parameters of membrane, permeant and vehicle compatibility and their interactions of relevance to membrane permeation. [ABSTRACT FROM AUTHOR]

Background Combined diffusion capacity of the lung for carbon monoxide (DL co ) and nitric oxide (DL no ) measurements allow for the estimation of pulmonary capillary blood volume (Vc) and alveolar membrane diffusion (Dm). The clinical usefulness of these measurements in pulmonary hypertension (PH) is unclear. Methods Combined DL co and DL no were measured in 290 consecutive patients with precapillary PH (pulmonary arterial hypertension (PAH), n = 153; pulmonary veno-occlusive disease (PVOD), n = 33; and chronic thromboembolic pulmonary hypertension (CTEPH), n = 104). Clinical correlates of Vc and Dm were assessed in a sub-group of PAH patients without comorbidities. Results PVOD patients compared with PAH and CTEPH patients displayed the lowest values of Vc (29.4 ± 16.8 ml vs 56.3 ± 26.5 ml vs 56.9 ± 26.2 ml, p < 0.01, respectively) and Dm (27.7 ± 11.6 ml/mm Hg/min vs 43.4 ± 14.8 ml/mm Hg/min vs 44.7 ± 17.7 ml/mm Hg/min, p < 0.01, respectively). The DL no /DL co ratio was highest in the PVOD group (5.82 ± 2.04 vs 4.95 ± 1.31 vs 5.16 ± 1.58, p < 0.05). In a sub-set of 69 PAH patients without comorbidities, Vc and Dm correlated significantly with functional capacity (6-minute walking distance, oxygen consumption) and New York Heart Association Functional Classification but not with invasive hemodynamics. Only Dm was significantly associated with survival. On receiver operating characteristic curve analysis, Vc, Dm, and DL no /DL co were not superior to DL co for discriminating PVOD from PAH or CTEPH. Conclusions PVOD patients display higher values of the DL no /DL co ratio compared with PAH and CTEPH, suggesting proportionally greater reduction in Vc relative to Dm. However, partitioning of diffusion failed to be more clinically relevant than conventional DL co for detection of PVOD. [ABSTRACT FROM AUTHOR]

Single-particle tracking photoactivated localization microscopy (sptPALM) has recently emerged as a powerful tool for high-density imaging and tracking of individual molecules in living cells. In this work, we have monitored and compared the diffusion dynamics of TGF-β type II receptor (TβRII) at high expression level using both traditional single-particle tracking (SPT) and sptPALM. The ligand-induced aggregation of TβRII oligomers was further indicated by sptPALM. Due to the capacity of distinguishing and tracking single molecules within diffraction limit, sptPALM outperforms traditional SPT by providing more accurate biophysical information. [ABSTRACT FROM AUTHOR]

Cell polarity refers to a functional spatial organization of proteins that is crucial for the control of essential cellular processes such as growth and division. To establish polarity, cells rely on elaborate regulation networks that control the distribution of proteins at the cell membrane. In fission yeast cells, a microtubule-dependent network has been identified that polarizes the distribution of signaling proteins that restricts growth to cell ends and targets the cytokinetic machinery to the middle of the cell. Although many molecular components have been shown to play a role in this network, it remains unknown which molecular functionalities are minimally required to establish a polarized protein distribution in this system. Here we show that a membrane-binding protein fragment, which distributes homogeneously in wild-type fission yeast cells, can be made to concentrate at cell ends by attaching it to a cytoplasmic microtubule end-binding protein. This concentration results in a polarized pattern of chimera proteins with a spatial extension that is very reminiscent of natural polarity patterns in fission yeast. However, chimera levels fluctuate in response to microtubule dynamics, and disruption of microtubules leads to disappearance of the pattern. Numerical simulations confirm that the combined functionality of membrane anchoring and microtubule tip affinity is in principle sufficient to create polarized patterns. Our chimera protein may thus represent a simple molecular functionality that is able to polarize the membrane, onto which additional layers of molecular complexity may be built to provide the temporal robustness that is typical of natural polarity patterns. [ABSTRACT FROM AUTHOR]

Diacylglycerols (DAGs) with unsaturated acyl chains play many important roles in biomembranes, such as a second messenger and activator for protein kinase C. In this study, three DAGs of distinctly different chain unsaturations (i.e. di16:0DAG (DPG), 16:0–18:1DAG (POG), and di18:1DAG (DOG)) are studied using atomistic MD simulation to compare their roles in the structure and dynamics of 16:0–18:1phosphatidylcholine (POPC) membranes. All three DAGs are able to produce the so-called ‘condensing effect’ in POPC membranes: decreasing area-per-lipid, and increasing acyl chain order and bilayer thickness. Our visual and quantitative analyses clearly show that DAG with unsaturated chains induce larger spacing between POPC headgroups, compared with DAG with saturated chains; this particular effect has long been hypothesized to be crucial for activating enzymes and receptors in cell membranes. DAGs with unsaturated chains are also located closer to the bilayer/aqueous interface than DPG and are more effective in slowing down lateral diffusion of molecules. We show that DAG molecules seek the “umbrella coverage” from neighboring phospholipid headgroups — similar to cholesterol. Unlike cholesterol, DAGs also hide their chains from water by laterally inserting their chains into the surrounding. Thus, acyl chains of DAG are more spread and disordered than those of PC due to the insertion. By calculating the potential of mean force (PMF) for POPC in POPC/DAG bilayers, we found that all three DAGs can significantly increase the free energy barrier for POPC to flip-flop, but only DAGs with unsaturated chains can additionally increase the free energy of POPC desorption. [ABSTRACT FROM AUTHOR]

Background: Reduced gas transfer in patients with pulmonary arterial hypertension (PAH) is traditionally attributed to remodeling and progressive loss of pulmonary arterial vasculature that results in decreased capillary blood volume available for gas exchange. Methods: We tested this hypothesis by determination of lung diffusing capacity (DL) and its components, the alveolar capillary membrane diffusing capacity (Dm) and lung capillary blood volume (Vc) in 28 individuals with PAH in comparison to 41 healthy individuals, and in 19 PAH patients over time. Using single breath simultaneous measure of diffusion of carbon monoxide (DLco) and nitric oxide (DLNO), DL and Dm were respectively determined, and Vc calculated. Dm and Vc were evaluated over time in relation to standard clinical indicators of disease severity, including brain natriuretic peptide (BNP), 6-minute walk distance (6MWD) and right ventricular systolic pressure (RVSP) by echocardiography. Results: Both DLCO and DLNO were reduced in PAH as compared to controls and the lower DL in PAH was due to loss of both Dm and Vc (all p < 0.01). While DLCO of PAH patients did not change over time, DLNO decreased by 24 ml/min/mmHg/year (p = 0.01). Consequently, Dm decreased and Vc tended to increase over time, which led to deterioration of the Dm/Vc ratio, a measure of alveolar-capillary membrane functional efficiency without changes in clinical markers. Conclusions: The findings indicate that lower than normal gas transfer in PAH is due to loss of both Dm and Vc, but that deterioration of Dm/Vc over time is related to worsening membrane diffusion. [ABSTRACT FROM AUTHOR]

Abstract: The separation and recovery of the major components contained in spent pickling effluents, that is, iron, zinc and HCl, is a key issue in the definition of sustainable treatments of this kind of industrial effluents. We propose a hybrid membrane process consisting first of a liquid membrane step that generates a raffinate highly concentrated in iron chloride and a mixture of HCl and zinc chlorides in the stripping phase. Second, diffusion or Donnan dialysis have been applied to the stripped solution in order to further separate the acidic stream in the diffusate from a solution containing zinc chlorides in dialyzate. Industrial effluents with an average composition of 1.2kmol/m3 of Zn, 1.6kmol/m3 of Fe, and 6.5kmol/m3 of HCl were previously used to analyse the kinetics of the metallic separation with the emulsion liquid membranes; this study reports the experimental and theoretical analysis of membrane dialysis of the stripped solution; the influence of operative variables such as concentration of HCl (0.5 to 3.0kmol/m3) and ZnCl2 (0.25 to 1.0kmol/m3), type of the anion-exchange membranes, and the basicity of the stripping agent on the dialysis flux and selectivity has been analysed. [Copyright &y& Elsevier]

Abstract: A novel two-stage reverse dialysis method has been developed for in vitro release testing of liposomal drug product with passive targeting characteristics. The first stage of the test is to mimic the circulation of liposomes in the body, whereas the second stage is to imitate the drug release process at the target. Buffer and surfactant solution were used during the first and second stages, respectively. For formulations containing high phase transition temperature lipids and high cholesterol content, no drug leakage was observed during the first stage of test. In the second stage, however, formulations with different compositions showed significant differences in terms of drug release rate, and discriminatory ability of the method was demonstrated. On comparing two different membrane diffusion techniques, dialysis and reverse dialysis methods, the reverse dialysis method showed significantly lower variation, and therefore is the preferred method. The developed in vitro release testing method should help to distinguish formulations with varied compositions for quality control testing purposes. This two-stage reverse dialysis method may pave the way to the development of more bio-relevant release testing methods for liposomal drug products. [Copyright &y& Elsevier]

Abstract: Molecular transport in confined spaces is of central importance to many traditional and emerging applications, such as gas separation and storage, catalytic and non-catalytic fluid–solid reactions and nanofluidics. The classical method to model the influence of solid–fluid particles collisions in transport comes from the early works of Knudsen and Smoluchowski, in which van-der-Walls interactions are neglected. While such assumption may be adequate for wide channels or high temperatures, it has been shown to lead to significant overprediction of the low-density diffusion coefficient in narrow pores at low and moderate temperatures, when compared to that obtained through molecular dynamics simulations. However, while molecular dynamics offers a much more accurate route for the calculation of diffusivities, its implementation and use is time-consuming and the Knudsen formulation remains preferred, particularly for interpretation of experimental permeation data. On the other hand, the oscillator model, developed in recent years in this laboratory, provides a straightforward method to estimate the low-density diffusivity, taking into account the dispersive forces exerted by the wall through rigorous statistical–mechanical considerations. The computational demand of the oscillator model is several orders of magnitude lower than that of a typical molecular dynamics run, though much greater than that of the simple Knudsen formulation. In order to facilitate its use, we provide several simple correlations for the fast estimation of the oscillator model-based diffusion coefficient for LJ fluids in simple geometries, as a function of the pore size and the LJ fluid–solid interaction parameters. Moreover, the parameter values for which the Knudsen equation supplies a reasonable estimation of the diffusion coefficient are presented for various degrees of accuracy. [Copyright &y& Elsevier]

Lipophilic fluorescent dyes have been used to trace neuronal connections because of their ability to diffuse laterally within nerve cell membranes. Given the hundreds to thousands of connections that a typical neuron makes with its neighbours, a diffusion-matched set of spectrally distinct dyes is desirable. To extend a set of these dyes to obtain six independent labels, we have characterized the properties of novel violet and near-infrared candidates. By combining two-photon and confocal microscopy all of these candidates can be imaged using a single Titanium Sapphire laser. Here we present measurements of the two-photon action cross-sections and diffusion properties of the dyes, using either the relative diffusion distance or fluorescence recovery after photobleaching techniques, and demonstrate six-colour neuronal tracing within the spinal cord and brain tissue. [ABSTRACT FROM AUTHOR]

Abstract: The uptake and diffusion of solvents across polymer membranes is important in controlled drug delivery, effects on drug uptake into, for example, infusion bags and containers, as well as transport across protective clothing. Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy has been used to monitor the effects of different solvents on the diffusion of a model compound, 4-cyanophenol (CNP) across silicone membrane and on the equilibrium concentration of CNP obtained in the membrane following diffusion. ATR-FTIR spectroscopic imaging of membrane diffusion was used to gain an understanding of when the boundary conditions applied to Fick’s second law, used to model the diffusion of permeants across the silicone membrane do not hold. The imaging experiments indicated that when the solvent was not taken up appreciably into the membrane, the presence of discrete solvent pools between the ATR crystal and the silicone membrane can affect the diffusion profile of the permeant. This effect is more significant if the permeant has a high solubility in the solvent. In contrast, solvents that are taken up into the membrane to a greater extent, or those where the solubility of the permeant in the vehicle is relatively low, were found to show a good fit to the diffusion model. As such these systems allow the ATR-FTIR spectroscopic approach to give mechanistic insight into how the particular solvents enhance permeation. The solubility of CNP in the solvent and the uptake of the solvent into the membrane were found to be important influences on the equilibrium concentration of the permeant obtained in the membrane following diffusion. In general, solvents which were taken up to a significant extent into the membrane and which caused the membrane to swell increased the diffusion coefficient of the permeant in the membrane though other factors such as solvent viscosity may also be important. [Copyright &y& Elsevier]

Single-particle tracking (SPT) applications have been growing rapidly in the field of cell biology, and in particular in neurobiology, as a means of unravelling the involvement of diffusion dynamics of neurotransmitter receptors and other synaptic proteins in the regulation of neuronal activity. Suitable probes and technological improvements make SPT more accessible than it used to be and open up broad applications in cellular biology. In this technical highlight, we give an overview of the experimental approach in SPT. The concepts and results in neurobiology have already been the object of detailed reviews. Here, we focus on a qualitative description of the implementation of SPT, from molecule labelling to acquisition, data treatment and analysis of protein diffusion properties. Constraints, limitations and future developments are discussed. [ABSTRACT FROM AUTHOR]

Recent experimental developments have led to a revision of the classical fluid mosaic model proposed by Singer and Nicholson more than 35 years ago. In particular, it is now well established that lipids and proteins diffuse heterogeneously in cell plasma membranes. Their complex motion patterns reflect the dynamic structure and composition of the membrane itself, as well as the presence of the underlying cytoskeleton scaffold and that of the extracellular matrix. How the structural organization of plasma membranes influences the diffusion of individual proteins remains a challenging, yet central, question for cell signaling and its regulation. Here we have developed a raft-associated glycosyl-phosphatidyl-inositol-anchored avidin test probe (Av-GPI), whose diffusion patterns indirectly report on the structure and dynamics of putative raft microdomains in the membrane of HeLa cells. Labeling with quantum dots (qdots) allowed high-resolution and long-term tracking of individual Av-GPI and the classification of their various diffusive behaviors. Using dual-color total internal reflection fluorescence (TIRF) microscopy, we studied the correlation between the diffusion of individual Av-GPI and the location of glycosphingolipid GM1-rich microdomains and caveolae. We show that Av-GPI exhibit a fast and a slow diffusion regime in different membrane regions, and that slowing down of their diffusion is correlated with entry in GM1-rich microdomains located in close proximity to, but distinct, from caveolae. We further show that Av-GPI dynamically partition in and out of these microdomains in a cholesterol-dependent manner. Our results provide direct evidence that cholesterol-/sphingolipid-rich microdomains can compartmentalize the diffusion of GPI-anchored proteins in living cells and that the dynamic partitioning raft model appropriately describes the diffusive behavior of some raft-associated proteins across the plasma membrane. [ABSTRACT FROM AUTHOR]

Abstract: This work studies the effect of two membrane-formation parameters, evaporation time and casting thickness, on the diffusive mass transport of organic solutes through an organic solvent nanofiltration (OSN) membrane. These parameters showed a coupled effect on the final membrane thickness, which was explained in terms of top-layer formation. In a concentration-driven dialysis, both parameters, as well as the resulting membrane thickness, had a significant effect on mass transport. Casting thickness had the greatest effect on membrane mass transport rates. Multivariate regression was used to model the dialysis process with acceptable fit. A representation of the membrane morphology was obtained from SEM pictures and used to formulate an alternative mechanistic mass-transport model. A resistance-circuit analogy was used to describe transport through the top and microporous layers, which also considered diffusion through the pores and the polymer for each layer. From the analyses of the models and considering that no differences in top-layer thickness were observed by SEM, it is concluded that membrane asymmetry, determined by the formation parameters, controls mass transport, rather than top-layer thickness. [Copyright &y& Elsevier]

Abstract: G protein-coupled receptors are known to form homo-and heteromers at the plasma membrane, but the molecular properties of these oligomers are relatively unknown. Here, we show a method that allows the diffusion of G protein-coupled receptors oligomers in the plasma membrane to be monitored in single cells by combining Bimolecular Fluorescence Complementation and Fluorescence Correlation Spectroscopy. With this approach we have measured, for the first time, the membrane diffusional characteristics of adenosine A1 and A2A receptor homo-and heterodimers in Chinese Hamster Ovary cells. Interestingly, both homodimers display similar diffusion co-efficients (D) when expressed in living cells (D =5.0 and 4.8×10−9 cm2/s, respectively) but the heterodimer formed by these receptors exhibit a significantly faster plasma membrane diffusion co-efficent (D =5.6×10−9 cm2/s) when compared to the adenosine A1 receptor tagged with the full-length yellow fluorescent protein (D =4.0×10−9 cm2/s). Overall, these results demonstrate differences in plasma membrane diffusion between adenosine receptor homo-and heterodimers, providing new insights into the molecular plasticity of G protein-coupled receptor oligomerization. [Copyright &y& Elsevier]

We sought to determine the influence of sildenafil on the diffusing capacity of the lungs for carbon monoxide (DLCO) and the components of DLCO (pulmonary capillary blood volume VC, and alveolar-capillary membrane conductance DM) at rest and following exercise with normoxia and hypoxia. This double-blind placebo-controlled, cross-over study included 14 healthy subjects (age = 33 +/- 11 years, ht = 181 +/- 8 cm, weight = 85 +/- 14 kg, BMI = 26 +/- 3 kg/m2, peak normoxic VO2 = 36 +/- 6 ml/kg, mean +/- SD). Subjects were randomized to placebo or 100 mg sildenafil 1 h prior to entering a hypoxic tent with an FiO2 of 12.5% for 90 min. DLCO, VC, and DM were assessed at rest, every 3 min during exercise, at peak exercise, and 10 and 30 min post exercise. Sildenafil attenuated the elevation in PAP at rest and during recovery with exposure to hypoxia, but pulmonary arterial pressure immediately post exercise was not different between sildenafil and placebo. Systemic 02 saturation and VO2peak did not differ between the two conditions. DLCO was not different between groups at any time point. VC was higher with exercise in the placebo group, and the difference in DM between sildenafil and placebo was significant only when corrected for changes in VC (DM/VC = 0.57 +/- 0.29 vs. 0.41 +/- 0.16, P = 0.04). These results suggest no effect of sildenafil on DLCO, but an improvement in DM when corrected for changes in VC during short-term hypoxic exposure with exercise. [ABSTRACT FROM AUTHOR]

Abstract: Fluorescence Recovery After Photobleaching (FRAP) using the confocal laser scanning microscope has become a standard method used to determine the diffusion coefficient and mobile fraction of cell surface proteins. A common experimental approach is to bleach a stripe on the cell surface and fit the ensuing FRAP curve to a 1D diffusion model. This model is derived from the time course of recovery to an infinitely long stripe bleached on an infinite flat plane. This choice of model dictates the use of a long bleach stripe. We demonstrate that, in the case of a long bleach stripe, the finite extent of the cell leads to significant errors in parameter estimation. We further show that these errors are reduced when a relatively small stripe is bleached. Unfortunately, diffusion to such a region is fundamentally two dimensional and therefore applying the 1D model of diffusion leads to significant errors. We derive an equation suitable for fitting to FRAP data acquired from small bleach regions and analyze its accuracy using simulated data. We propose that the use of a small bleach region along with a two dimensional diffusion model is the ideal protocol for cell surface FRAP. [Copyright &y& Elsevier]

The light-harvesting complex II (LHCII) is the main energy absorber for photosynthesis in green plants, and its translocation between photosystems I and II is the primary means of energy redistribution between them. Using single-particle tracking, we performed the first measurement of the mobility of LHCII in the photosynthetic membranes in both the nonphosphorylated and the phosphorylated (P-LHCII) conformations. These are part of an important, reversible, energy re-equilibration process called the state transition. We found that the population of P-LHCII in unappressed membranes is more mobile than the population of non-P-LHCII from the same regions. [ABSTRACT FROM AUTHOR]

Abstract: The rotating dialysis cell consisting of a donor and an acceptor compartment with volumes of 10 and 1000ml, respectively, separated by a dialysis membrane is proposed as an in vitro model potentially useful for simulation of the events influencing drug residence time in the knee joint cavity after intra-articular instillation. The purpose of this study was to characterize the rotating dialysis cell model with respect to basic model and drug related factors affecting the rate of drug appearance in the acceptor phase after initial instillation of the solutes into the donor cell. A total of 15 model compounds were included in the study and it was revealed that the transport processes were governed by (i) the volume of the donor solution and (ii) the molecular weight of the diffusants. A relationship between the donor volume-independent permeability coefficient and the molecular weight of the diffusants has been established. Additionally, the model was robust with release kinetics being insensitive to changes in pH, ionic strength, viscosity of the release medium, and revolution speed of the donor cell. The characteristics of the rotating dialysis cell model suggest that it may be a useful tool in the design of innovative depot injectables in the area of local joint delivery. [Copyright &y& Elsevier]