A functioning artificial secretory cell

We present an amperometric study of content release from individual vesicles in an artificial secretory cell designed with the minimal components required to carry out exocytosis. Here, the membranes of the cell and vesicles are substituted for protein-free giant and large unilamellar vesicles respectively. In replacement of the SNARE-complex, the cell model was equipped with an analog composed of complimentary DNA constructs. The DNA constructs hybridize in a zipper-like fashion to bring about docking of the artificial secretory vesicles and following the addition of Ca2+ artificial exocytosis was completed. Exocytotic events recorded from the artificial cell closely approximate exocytosis in live cells. The results together with simulations of vesicular release demonstrate that the molecular flux in this model is attenuated and we suggest that this is the result of restricted diffusion through a semi-stable fusion pore or a partitioning of the signalling molecule out of the fused vesicle membrane

The use of multiplex platforms for absolute and relative protein quantification of clinical material

Abstract When introducing multiplex platforms to measure protein content in precious clinical material there is an increased risk of cross reactivity, loss of sensitivity as well as accuracy. In this paper, four multiplex platforms and one singleplex platform were compared by running pre- and post-treatment plasma samples from CML patients. We found a variation of absolute protein concentrations between platforms. For some of the analytes and platforms, relative differences between pre- and post-treatment samples correlated. We conclude that absolute concentrations measured by different platforms should be compared with caution and comparing relative differences could be more accurate.Peer reviewe

Plasma proteomics in CML patients before and after initiation of tyrosine kinase inhibitor therapy reveals induced Th1 immunity and loss of angiogenic stimuli

Background and aims: The simultaneous measurement of many proteins is now possible using multiplex assays. In this pilot study we investigated a total of 124 proteins in plasma from chronic myeloid leukemia (CML) patients with the purpose of identifying proteins that are differently expressed at diagnosis and after tyrosine kinase inhibitor (TKI) treatment initiation. Methods: Samples were taken from 14 CML patients at diagnosis and after three months of TKI treatment (imatinib or dasatinib). Samples were analyzed by Mesoscale Discovery, Myriad RBM MAP technology and Olink Proseek. Results: Multiple plasma proteins were differentially expressed before and after initiation of TKI therapy. Protein patterns demonstrated a possible shift towards Th1-immunity and reduced angiogenic stimuli. Further, some plasma proteins were identified that can be of potential interest to study further for biologic, prognostic or therapeutic significance such as E-selectin, uPAR, growth hormone and carbonic anhydrase IX. Conclusions: Plasma proteomics seems feasible and useful in CML patients, both for studying patterns of protein expression and for identifying single proteins differentially expressed before and after treatment. Plasma proteomics may be useful to map disease activity and biological processes. Hence, plasma proteomics can be used to understand drug mechanisms and treatment responses in CML. (C) 2016 Elsevier Ltd. All rights reserved.Peer reviewe

Waveguide structure

A waveguide structure for evanescent wave microscopy and/or spectroscopy, comprising an optically transparent core layer, a lower dielectric cladding layer and an upper dielectric cladding layer arranged on opposite sides of the core layer. The core layer has a refractive index higher than the refractive indices of the cladding layers. The upper cladding layer is made of an organic material. A sample well is arranged on an upper surface of the core layer formed by a cavity in the upper cladding layer, the sample well being adapted to contain a sample medium with one or more sample objects. The core layer is made of a first dielectric inorganic material, and the upper cladding layer has a refractive index which closely matches the refractive index of the sample medium. A method for manufacturing such waveguide structure, and a measurement system comprising the waveguide structure are also disclosed

Increased Level of Myeloid-Derived Suppressor Cells, Programmed Death Receptor Ligand 1/Programmed Death Receptor 1, and Soluble CD25 in Sokal High Risk Chronic Myeloid Leukemia

Peer reviewe

Increased Level of Myeloid-Derived Suppressor Cells, Programmed Death Receptor Ligand 1/Programmed Death Receptor 1, and Soluble CD25 in Sokal High Risk Chronic Myeloid Leukemia

Peer reviewe

DNA-Controlled Lipid-Membrane Fusion

Membrane fusion is essential for nerve-cell communication, for protein transport between cell organelles and the cell-membrane and for enabling the merger between virus and host membranes during virus infection. We have demonstrated that short DNA oligonucleotides, membrane-attached via CH in an orientation that mimics the overall zipperlike architecture of fusion-inducing proteins, induce fusion of both suspended vesicles and vesicles site-specifically tethered to SLBs. The site-specific surface-based assay is attractive for membrane-protein array applications.Model systems for membrane fusion have been generated from lipid vesicles, which are artificial spherical cell membranes encapsulating liquid compartments. In the bulk assay, two vesicle populations were decorated with complementary DNA strands which hybridize in a zipper-like fashion to bring about membrane fusion. The DNA strands were anchored in the lipid membranes of the vesicles via a hydrophobic moiety, cholesterol (CH), covalently attached at one end of the DNA strands. The lipid rearrangements taking place as a consequence of the forced bilayer contact and subsequent fusion were investigated using fluorescence resonance energy transfer (FRET) between donor and acceptor dyes. Total lipid mixing, inner leaflet mixing as well as content mixing was monitored. In order to design a DNA zipper with improved fusion properties, we have assessed how parameters such as length of the DNA strands, anchoring strategy and DNA surface coverage affect the ability of DNA to induce fusion. The results reveal that the use of two CH anchors is essential to prevent complementary DNA strands from shuttling between differently modified lipid vesicles. A surface coverage of 6-13 DNA strands per lipid vesicle was a precondition for efficient fusion, whereas fusion was insensitive to DNA length within the tested range.Implementation of the DNA-controlled fusion concept into a site-specific surface-based assay, by fusing the vesicles to a SLB, brings the concept towards site-specific delivery of membrane constituents and thus further towards the realization of a protein array bio-sensor. The geometry of the surface-based system resembles that of native vesicles fusing with the cell membrane better than do the vesicle-vesicle fusion assay. The surface-based fusion assay also provides details and heterogeneities of the fusing vesicle population, as it allowed us to study single vesicle fusion events. From the results we learn that fusion is observed for a specific range of 10-16 DNA strands per vesicle, and from studies of the diffusion of the tethered vesicles prior to fusion, together with the vesicle docking time prior to fusion, a possible scenario for the zipper-DNA induced vesicle-SLB fusion machinery is proposed. Future applications and upcoming projects are also discussed

Lipid Vesicle Fusion: Investigation, Generation and Manipulation of Cell-Membrane Mimics

Membrane fusion is essential for nerve-cell communication, for protein transport between cell organelles and the cell-membrane and for enabling the merger between virus and host membranes during virus infection. In this work, cell-membrane mimics were constructed and evaluated as models for studies of the membrane-fusion process. As a mimic of SNARE-proteins, known to induce fusion in secretory cells (exocytosis), short cholesterol-tagged DNA strands were used to facilitate fusion. The DNA strands were proven efficient as SNARE-protein substitutes in terms of bringing lipid vesicle fusion and content release about. This thus enabled reductionist and protein-free studies of some of the mechanisms behind lipid vesicle fusion. In particular, the fast kinetics of vesicle-content release upon fusion was possible to resolve using amperometry. The model system designed to be compatible with amperometric recordings generated data that could be directly compared with amperometric recordings from live cells. In combination with theoretical modeling, this has led us to suggest that the opening of the fusion pore is the rate-limiting step for content release in a protein-free system. In combination with the ability to systematically alter additional parameters, such as lipid composition, this model system may help resolving some of the still unanswered questions regarding the molecular mechanisms that control the exocytosis process.As a second approach, lipid vesicle fusion was used as a new means to form planar cell-membrane mimics on solid supports. It is demonstrated that such supported lipid bilayers (SLBs) could be made with complex lipid compositions that are generally prevented when alternative SLB-formation methods are used. In particular, successful formation of SLBs containing native cell membrane components was demonstrated. Furthermore, by forming such SLBs in microfluidic systems, spatial manipulation of cell-membrane-associated molecules was demonstrated. This, in turn, points towards the exciting possibility to both enrich and separate membrane proteins while in their native environment, potentially offering a new way to study this biologically and pharmacologically very important class of biomolecules

Lipid Vesicle Fusion: Investigation, Generation and Manipulation of Cell-Membrane Mimics

Membrane fusion is essential for nerve-cell communication, for protein transport between cell organelles and the cell-membrane and for enabling the merger between virus and host membranes during virus infection. In this work, cell-membrane mimics were constructed and evaluated as models for studies of the membrane-fusion process. As a mimic of SNARE-proteins, known to induce fusion in secretory cells (exocytosis), short cholesterol-tagged DNA strands were used to facilitate fusion. The DNA strands were proven efficient as SNARE-protein substitutes in terms of bringing lipid vesicle fusion and content release about. This thus enabled reductionist and protein-free studies of some of the mechanisms behind lipid vesicle fusion. In particular, the fast kinetics of vesicle-content release upon fusion was possible to resolve using amperometry. The model system designed to be compatible with amperometric recordings generated data that could be directly compared with amperometric recordings from live cells. In combination with theoretical modeling, this has led us to suggest that the opening of the fusion pore is the rate-limiting step for content release in a protein-free system. In combination with the ability to systematically alter additional parameters, such as lipid composition, this model system may help resolving some of the still unanswered questions regarding the molecular mechanisms that control the exocytosis process.As a second approach, lipid vesicle fusion was used as a new means to form planar cell-membrane mimics on solid supports. It is demonstrated that such supported lipid bilayers (SLBs) could be made with complex lipid compositions that are generally prevented when alternative SLB-formation methods are used. In particular, successful formation of SLBs containing native cell membrane components was demonstrated. Furthermore, by forming such SLBs in microfluidic systems, spatial manipulation of cell-membrane-associated molecules was demonstrated. This, in turn, points towards the exciting possibility to both enrich and separate membrane proteins while in their native environment, potentially offering a new way to study this biologically and pharmacologically very important class of biomolecules

DNA-Controlled Lipid-Membrane Fusion

Membrane fusion is essential for nerve-cell communication, for protein transport between cell organelles and the cell-membrane and for enabling the merger between virus and host membranes during virus infection. We have demonstrated that short DNA oligonucleotides, membrane-attached via CH in an orientation that mimics the overall zipperlike architecture of fusion-inducing proteins, induce fusion of both suspended vesicles and vesicles site-specifically tethered to SLBs. The site-specific surface-based assay is attractive for membrane-protein array applications.Model systems for membrane fusion have been generated from lipid vesicles, which are artificial spherical cell membranes encapsulating liquid compartments. In the bulk assay, two vesicle populations were decorated with complementary DNA strands which hybridize in a zipper-like fashion to bring about membrane fusion. The DNA strands were anchored in the lipid membranes of the vesicles via a hydrophobic moiety, cholesterol (CH), covalently attached at one end of the DNA strands. The lipid rearrangements taking place as a consequence of the forced bilayer contact and subsequent fusion were investigated using fluorescence resonance energy transfer (FRET) between donor and acceptor dyes. Total lipid mixing, inner leaflet mixing as well as content mixing was monitored. In order to design a DNA zipper with improved fusion properties, we have assessed how parameters such as length of the DNA strands, anchoring strategy and DNA surface coverage affect the ability of DNA to induce fusion. The results reveal that the use of two CH anchors is essential to prevent complementary DNA strands from shuttling between differently modified lipid vesicles. A surface coverage of 6-13 DNA strands per lipid vesicle was a precondition for efficient fusion, whereas fusion was insensitive to DNA length within the tested range.Implementation of the DNA-controlled fusion concept into a site-specific surface-based assay, by fusing the vesicles to a SLB, brings the concept towards site-specific delivery of membrane constituents and thus further towards the realization of a protein array bio-sensor. The geometry of the surface-based system resembles that of native vesicles fusing with the cell membrane better than do the vesicle-vesicle fusion assay. The surface-based fusion assay also provides details and heterogeneities of the fusing vesicle population, as it allowed us to study single vesicle fusion events. From the results we learn that fusion is observed for a specific range of 10-16 DNA strands per vesicle, and from studies of the diffusion of the tethered vesicles prior to fusion, together with the vesicle docking time prior to fusion, a possible scenario for the zipper-DNA induced vesicle-SLB fusion machinery is proposed. Future applications and upcoming projects are also discussed