| 1. |
- Jungersted, Jakob Mutanu, et al.
(författare)
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In vivo studies of aquaporins 3 and 10 in human stratum corneum.
- 2013
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Ingår i: Archives of dermatological research. - : Springer Science and Business Media LLC. - 1432-069X .- 0340-3696. ; 305:8, s. 699-704
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporins (AQPs) constitute one family of transmembrane proteins facilitating transport of water across cell membranes. Due to their specificity, AQPs have a broad spectrum of physiological functions, and for keratinocytes there are indications that these channel proteins are involved in cell migration and proliferation with consequences for the antimicrobial defense of the skin. AQP3 and AQP10 are aqua-glyceroporins, known to transport glycerol as well as water. AQP3 is the predominant AQP in human skin and has previously been demonstrated in the basal layer of epidermis in normal human skin, but not in stratum corneum (SC). AQP10 has not previously been identified in human skin. Previous studies have demonstrated the presence of AQP3 and AQP10 mRNA in keratinocytes. In this study, our aim was to investigate if these aquaporin proteins were actually present in human SC cells. This can be seen as a first step toward elucidating the possible functional role of AQP3 and AQP10 in SC hydration. Specifically we investigate the presence of AQP3 and AQP10 in vivo in human SC using "minimal-invasive" technique for obtaining SC samples. SC samples were obtained from six healthy volunteers. Western blotting and immunohistochemistry were used to demonstrate the presence of AQP3 as well as AQP10. The presence of AQP3 and AQP10 was verified by Western blotting, allowing for detection of proteins by specific antibodies. Applying immunohistochemistry, cell-like structures in the shape of corneocytes were identified in all samples by AQP3 and AQP10 antibodies. In conclusion, identification of AQP3 and AQP10 protein in SC in an in vivo model is new. Together with the new "minimal-invasive" method for SC collection presented, this opens for new possibilities to study the role of AQPs in relation to function of the skin barrier.
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| 2. |
- Bjørkskov, Frederik Bühring, et al.
(författare)
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Purification and functional comparison of nine human Aquaporins produced in Saccharomyces cerevisiae for the purpose of biophysical characterization
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- The sparse number of high-resolution human membrane protein structures severely restricts our comprehension of molecular physiology and ability to exploit rational drug design. In the search for a standardized, cheap and easily handled human membrane protein production platform, we thoroughly investigated the capacity of S. cerevisiae to deliver high yields of prime quality human AQPs, focusing on poorly characterized members including some previously shown to be difficult to isolate. Exploiting GFP labeled forms we comprehensively optimized production and purification procedures resulting in satisfactory yields of all nine AQP targets. We applied the obtained knowledge to successfully upscale purification of histidine tagged human AQP10 produced in large bioreactors. Glycosylation analysis revealed that AQP7 and 12 were O-glycosylated, AQP10 was N-glycosylated while the other AQPs were not glycosylated. We furthermore performed functional characterization and found that AQP 2, 6 and 8 allowed flux of water whereas AQP3, 7, 9, 10, 11 and 12 also facilitated a glycerol flux. In conclusion, our S. cerevisiae platform emerges as a powerful tool for isolation of functional, difficult-To-express human membrane proteins suitable for biophysical characterization.
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| 3. |
- Gotfryd, Kamil, et al.
(författare)
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Human adipose glycerol flux is regulated by a pH gate in AQP10
- 2018
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Obesity is a major threat to global health and metabolically associated with glycerol homeostasis. Here we demonstrate that in human adipocytes, the decreased pH observed during lipolysis (fat burning) correlates with increased glycerol release and stimulation of aquaglyceroporin AQP10. The crystal structure of human AQP10 determined at 2.3 Å resolution unveils the molecular basis for pH modulation-an exceptionally wide selectivity (ar/R) filter and a unique cytoplasmic gate. Structural and functional (in vitro and in vivo) analyses disclose a glycerol-specific pH-dependence and pinpoint pore-lining His80 as the pH-sensor. Molecular dynamics simulations indicate how gate opening is achieved. These findings unravel a unique type of aquaporin regulation important for controlling body fat mass. Thus, targeting the cytoplasmic gate to induce constitutive glycerol secretion may offer an attractive option for treating obesity and related complications.
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| 4. |
- Habel, Joachim, et al.
(författare)
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Selecting analytical tools for characterization of polymersomes in aqueous solution
- 2015
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Ingår i: RSC Advances. - : Royal Society of Chemistry (RSC). - 2046-2069. ; 5:97, s. 79924-79946
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Forskningsöversikt (refereegranskat)abstract
- Selecting the appropriate analytical methods for characterizing the assembly and morphology of polymer-based vesicles, or polymersomes are required to reach their full potential in biotechnology. This work presents and compares 17 different techniques for their ability to adequately report size, lamellarity, elastic properties, bilayer surface charge, thickness and polarity of polybutadiene-polyethylene oxide (PB-PEO) based polymersomes. The techniques used in this study are broadly divided into scattering techniques, visualization methods, physical and electromagnetical manipulation and sorting/purification. Of the analytical methods tested, Cryo-transmission electron microscopy and atomic force microscopy (AFM) turned out to be advantageous for polymersomes with smaller diameter than 200 nm, whereas confocal microscopy is ideal for diameters >400 nm. Polymersomes in the intermediate diameter range can be characterized using freeze fracture Cryo-scanning electron microscopy (FF-Cryo-SEM) and nanoparticle tracking analysis (NTA). Small angle X-ray scattering (SAXS) provides reliable data on bilayer thickness and internal structure, Cryo-TEM on multilamellarity. Taken together, these tools are valuable for characterizing polymersomes per se but the comparative overview is also intended to serve as a starting point for selecting methods for characterizing polymersomes with encapsulated compounds or polymersomes with incorporated biomolecules (e.g. membrane proteins).
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| 5. |
- Hey, Tobias, et al.
(författare)
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Evaluation of direct membrane filtration and direct forward osmosis as concepts for compact and energy-positive municipal wastewater treatment
- 2018
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Ingår i: Environmental Technology. - : Informa UK Limited. - 0959-3330 .- 1479-487X. ; 39:3, s. 264-276
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Tidskriftsartikel (refereegranskat)abstract
- Municipal wastewater treatment commonly involves mechanical, biological and chemical treatment steps to protect humans and the environment from adverse effects. Membrane technology has gained increasing attention as an alternative to conventional wastewater treatment due to increased urbanization. Among the available membrane technologies, microfiltration (MF) and forward osmosis (FO) have been selected for this study due to their specific characteristics, such as compactness and efficient removal of particles. In this study, two treatment concepts were evaluated with regard to their specific electricity, energy and area demands. Both concepts would fulfil the Swedish discharge demands for small- and medium-sized wastewater treatment plants at full scale: (1) direct MF and (2) direct FO with seawater as the draw solution. The framework of this study is based on a combination of data obtained from bench- and pilot-scale experiments applying direct MF and FO, respectively. Additionally, available complementary data from a Swedish full-scale wastewater treatment plant and the literature were used to evaluate the concepts in depth. The results of this study indicate that both concepts are net positive with respect to electricity and energy, as more biogas can be produced compared to that using conventional wastewater treatment. Furthermore, the specific area demand is significantly reduced. This study demonstrates that municipal wastewater could be treated in a more energy- and area-efficient manner with techniques that are already commercially available and with future membrane technology.
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| 6. |
- Hey, Tobias, et al.
(författare)
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Influences of mechanical pretreatment on the non-biological treatment of municipal wastewater by forward osmosis
- 2017
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Ingår i: Environmental Technology. - : Informa UK Limited. - 0959-3330 .- 1479-487X. ; 38:18, s. 2295-2304
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Tidskriftsartikel (refereegranskat)abstract
- Municipal wastewater treatment involves mechanical, biological and chemical treatment steps for protecting the environment from adverse effects. The biological treatment step consumes the most energy and can create greenhouse gases. This study investigates municipal wastewater treatment without the biological treatment step, including the effects of different pretreatment configurations, for example, direct membrane filtration before forward osmosis. Forward osmosis was tested using raw wastewater and wastewater subjected to different types of mechanical pretreatment, for example, microsieving and microfiltration permeation, as a potential technology for municipal wastewater treatment. Forward osmosis was performed using Aquaporin Inside™ and Hydration Technologies Inc. (HTI) membranes with NaCl as the draw solution. Both types of forward osmosis membranes were tested in parallel for the different types of pretreated feed and evaluated in terms of water flux and solute rejection, that is, biochemical oxygen demand (BOD7) and total and soluble phosphorus contents. The Aquaporin and HTI membranes achieved a stable water flux with rejection rates of more than 96% for BOD7 and total and soluble phosphorus, regardless of the type of mechanical pretreated wastewater considered. This result indicates that forward osmosis membranes can tolerate exposure to municipal waste water and that the permeate can fulfil the Swedish discharge limits.
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| 7. |
- Hey, Tobias, et al.
(författare)
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The effects of physicochemical wastewater treatment operations on forward osmosis
- 2017
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Ingår i: Environmental Technology. - : Informa UK Limited. - 0959-3330 .- 1479-487X. ; 38:17, s. 2130-2142
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Tidskriftsartikel (refereegranskat)abstract
- Raw municipal wastewater from a full-scale wastewater treatment plant was physicochemically pretreated in a large pilot-scale system comprising coagulation, flocculation, microsieve and microfiltration operated in various configurations. The produced microsieve filtrates and microfiltration permeates were then concentrated using forward osmosis (FO). Aquaporin InsideTM FO membranes were used for both the microsieve filtrate and microfiltration permeates, and Hydration Technologies Inc.–thin-film composite membranes for the microfiltration permeate using only NaCl as the draw solution. The FO performance was evaluated in terms of the water flux, water flux decline and solute rejections of biochemical oxygen demand, and total and soluble phosphorus. The obtained results were compared with the results of FO after only mechanical pretreatment. The FO permeates satisfied the Swedish discharge demands for small and medium-sized wastewater treatment plants. The study demonstrates that physicochemical pretreatment can improve the FO water flux by up to 20%. In contrast, the solute rejection decreases significantly compared to the FO-treated wastewater with mechanical pretreatment.
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| 8. |
- Palanco, Marta Espina, et al.
(författare)
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Tuning biomimetic membrane barrier properties by hydrocarbon, cholesterol and polymeric additives
- 2018
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Ingår i: Bioinspiration and Biomimetics. - : IOP Publishing. - 1748-3182 .- 1748-3190. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- The barrier properties of cellular membranes are increasingly attracting attention as a source of inspiration for designing biomimetic membranes. The broad range of potential technological applications makes the use of lipid and lately also polymeric materials a popular choice for constructing biomimetic membranes, where the barrier properties can be controlled by the composition of the membrane constituent elements. Here we investigate the membrane properties reported by the light-induced proton pumping activity of bacteriorhodopsin (bR) reconstituted in three vesicle systems of different membrane composition. Specifically we quantify how the resulting proton influx and efflux rates are influenced by the membrane composition using a variety of membrane modulators. We demonstrate that by adding hydrocarbons to vesicles with reconstituted bR formed from asolectin lipids the resulting transmembrane proton fluxes changes proportional to the carbon chain length when compared against control. We observe a similar proportionality in single-component 1,2-Dioleoyl-sn-glycero-3-phosphocholine model membranes when using cholesterol. Lastly we investigate the effects of adding the amphiphilic di-block co-polymer polybutadiene-polyethyleneoxide (PB12-PEO10) to phospholipid membranes formed from 1,2-Dioleoyl-sn-glycero-3-phosphocholine, 1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine, and 1,2-Dioleoyl-sn-glycero-3-phosphatidylserine. The proton pumping activity of bR (measured as a change in extra-vesicular pH) in mixed lipid/PB12-PEO10 lipid systems is up to six-fold higher compared to that observed for bR containing vesicles made from PB12-PEO10 alone. Interestingly, bR inserts with apparent opposite orientation in pure PB12-PEO10 vesicles as compared to pure lipid vesicles. Addition of equimolar amounts of lipids to PB12-PEO10 results in bR orientation similar to that observed for pure lipids. In conclusion our results show how the barrier properties of the membranes can be controlled by the composition of the membrane. In particular the use of mixed lipid-polymer systems may pave the way for constructing biomimetic membranes tailored for optimal properties in various applications including drug delivery systems, biosensors and energy conservation technology.
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| 9. |
- Plasencia, Ines, et al.
(författare)
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Structure and Stability of the Spinach Aquaporin SoPIP2;1 in Detergent Micelles and Lipid Membranes
- 2011
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:2
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Tidskriftsartikel (refereegranskat)abstract
- Background: SoPIP2;1 constitutes one of the major integral proteins in spinach leaf plasma membranes and belongs to the aquaporin family. SoPIP2;1 is a highly permeable and selective water channel that has been successfully overexpressed and purified with high yields. In order to optimize reconstitution of the purified protein into biomimetic systems, we have here for the first time characterized the structural stability of SoPIP2;1. Methodology/Principal Finding: We have characterized the protein structural stability after purification and after reconstitution into detergent micelles and proteoliposomes using circular dichroism and fluorescence spectroscopy techniques. The structure of SoPIP2;1 was analyzed either with the protein solubilized with octyl-beta-D-glucopyranoside (OG) or reconstituted into lipid membranes formed by E. coli lipids, diphytanoylphosphatidylcholine (DPhPC), or reconstituted into lipid membranes formed from mixtures of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS), and ergosterol. Generally, SoPIP2;1 secondary structure was found to be predominantly a-helical in accordance with crystallographic data. The protein has a high thermal structural stability in detergent solutions, with an irreversible thermal unfolding occurring at a melting temperature of 58 degrees C. Incorporation of the protein into lipid membranes increases the structural stability as evidenced by an increased melting temperature of up to 70 degrees C. Conclusion/Significance: The results of this study provide insights into SoPIP2;1 stability in various host membranes and suggest suitable choices of detergent and lipid composition for reconstitution of SoPIP2;1 into biomimetic membranes for biotechnological applications.
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| 10. |
- Thuvander, Johan, et al.
(författare)
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Characterization of Irreversible Fouling after Ultrafiltration of Thermomechanical Pulp Mill Process Water
- 2018
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Ingår i: Journal of Wood Chemistry and Technology. - : Informa UK Limited. - 0277-3813 .- 1532-2319. ; 38:3, s. 276-285
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Tidskriftsartikel (refereegranskat)abstract
- Large volumes of wastewater with dissolved wood components are treated in wastewater treatment plants at thermomechanical pulp mills. It has been shown previously that hemicelluloses in these wastewater streams can be recovered by membrane filtration. A serious obstacle when treating lignocellulose process streams is fouling of the membranes. Fouling not only increases operating costs but also reduces the operating time of the membrane plant. When optimizing the membrane cleaning method, it is important to know which compounds cause the fouling. In this work fouling of an ultrafiltration membrane was studied. The fouling propensity of untreated process water and microfiltrated process water was compared. Fouled membranes were analyzed using scanning electron microscopy and attenuated total reflection Fourier transform infrared spectrometry. Acid hydrolysis of membranes exposed to untreated process water and microfiltration permeate revealed that 508 mg/m2 and 37 mg/m2 of polysaccharides, respectively, remained on the membranes even after alkaline cleaning.
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| 11. |
- Truelsen, Sigurd Friis, et al.
(författare)
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The role of water coordination in the pH-dependent gating of hAQP10
- 2022
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736. ; 1864:1
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Tidskriftsartikel (refereegranskat)abstract
- Human aquaporin 10 (hAQP10) is an aquaglyceroporin that assists in maintaining glycerol flux in adipocytes during lipolysis at low pH. Hence, a molecular understanding of the pH-sensitive glycerol conductance may open up for drug development in obesity and metabolically related disorders. Control of hAQP10-mediated glycerol flux has been linked to the cytoplasmic end of the channel, where a unique loop is regulated by the protonation status of histidine 80 (H80). Here, we performed unbiased molecular dynamics simulations of three protonation states of H80 to unravel channel gating. Strikingly, at neutral pH, we identified a water coordination pattern with an inverted orientation of the water molecules in vicinity of the loop. Protonation of H80 results in a more hydrophobic loop conformation, causing loss of water coordination and leaving the pore often dehydrated. Our results indicate that the loss of such water interaction network may be integral for the destabilization of the loop in the closed configuration at low pH. Additionally, a residue unique to hAQP10 (F85) reveals structural importance by flipping into the channel in correlation with loop movements, indicating a loop-stabilizing role in the closed configuration. Taken together, our simulations suggest a unique gating mechanism combining complex interaction networks between water molecules and protein residues at the loop interface. Considering the role of hAQP10 in adipocytes, the detailed molecular insights of pH-regulation presented here will help to understand glycerol pathways in these cells and may assist in drug discovery for better management of human adiposity and obesity.
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