Oxygen Supplementing, Biocompatible Outer Membranes for Enhanced Performance of Implantable Glucose Sensors

Lack of linearity and sensitivity, oxygen dependence, biofouling and tissue inflammation hinder the development of implantable biosensors for continuous monitoring of glucose. Herein, we report the development of stacked outer membranes based on LBL/PVA hydrogels that improve sensor sensitivity, linearity, oxygen independence and counter biofouling and inflammation. While the inner LBL membrane affords tunable diffusivity, the outer PVA is capable of releasing anti-inflammatory drugs/tissue response modifying agents to counter acute and chronic inflammation, and to induce neo-angiogenesis at the implant site. Sensors were fabricated by immobilizing GOx enzyme on top of 50 μm platinum wires, followed by deposition of stacked LBL/PVA hydrogel membranes. The response of the sensors at 0.7V to various glucose concentrations was studied. Michelis-Menten analysis was performed to quantify sensor performance in terms of linearity and oxygen dependence. The interplay between sensor performance and inward glucose diffusivity was elucidated using (i) various LBL membranes and (ii) various freeze-thaw (FT) cycles of PVA. Incorporation of LBL/PVA stacked membranes resulted in an 8 fold increase in sensor linearity and a 9 fold decrease in oxygen dependence compared to controls. The enhancement in the sensor performance is attributed to (i) the oxygen storing capability of PVA hydrogel due to the formation of hydrophobic domains during its freezing/ thawing employed for its physical crosslinking and (ii) regulation of glucose flux by the inner LBL membrane. Such membranes offer significant advantages over presently available outer membranes in lieu of (i) their ability to control inflammation, (ii) their modulus that closely matches that of subcutaneous human tissue, (iii) non-necessity of reactive chemical crosslinking agents, (iv) tunable sensitivity and (v) supplemental storage of oxygen.

Lipid-dependent topogenesis and function of membrane proteins

Membranes are essential for the integrity and function of the cell. The collective property of the lipid bilayer is critical in providing an optimal functioning environment for membrane proteins. The simple yet well-characterized bacterium Escherichia coli serves an ideal model system to study the function of specific lipids since its lipid content can be easily manipulated. The most abundant lipid in E. coli membrane is phosphatidylethanolamine (PE, 70-80%). A PE-lacking E. coli mutant displays a complex mixture of deficient phenotypes, suggesting a profound role for PE in different aspects of cell function. A novel role of PE as a topological and functional determinant for membrane proteins has been established using lactose permease (LacY) as a model protein. PE is found to be required for energy-dependent uphill transport process of LacY. In PE-lacking membranes, LacY undergoes a dramatic conformational change, and the first half of the protein adopts an inverted topology with respect to the bilayer plane. ^ The work reported here was initiated to understand the molecular properties of lipids that enable their function as topological and functional determinants for membrane proteins. A glycolipid, monoglucosyldiacylglycerol (MGlcDAG) which shares physicochemical similarities with PE, was introduced to PE-lacking E. coli membranes. The introduction of MGlcDAG suppresses many of the PE-deficient phenotypes, and in particular supports the function and native topology of LacY. ^ The lipid-sensitive topogenic signals encoded in the amino acid sequence of LacY were also identified. Native LacY adopts an inverted topology when synthesized without PE, but mutation of specific acidic residues in the cytoplasmic extra-membrane domains can prevent this inversion and supports a native topological organization of LacY in PE-lacking membranes. These results suggest that it is the interplay between the collective charge properties of the lipid bilayer and extra-membrane loops of protein that determines the final orientation of transmembrane domains. By comparing the similarities as well as differences between these two lipids, we established how specific physical and chemical properties of lipids influence various cell functions and elucidated the molecular basis for the novel role of lipids in determining membrane protein topology. ^

In vivo zippering of inner and outer mitochondrial membranes by a stable translocation intermediate

It was previously assumed that the import of cytoplasmically synthesized precursor proteins into mitochondria occurs through a single structure spanning both outer and inner membranes at contact sites. Based on recent findings, however, the two membranes appear to contain independent translocation elements that reversibly cooperate during protein import. This feature makes it difficult to generate a means of isolating a fully integrated and functional translocation complex. To study these independent translocases in vitro and in vivo, we have constructed a chimeric protein consisting of an N-terminal authentic mitochondrial precursor (delta1-pyrroline-5-carboxylate dehydrogenase) linked, through glutathione S-transferase, to IgG binding domains derived from staphylococcal protein A. This construct becomes trapped en route to the matrix, spanning both outer and inner membranes in such a way that the entire signal-less delta1-pyrroline-5-carboxylate dehydrogenase moiety reaches the matrix, while only the folded protein A domain remains outside. During in vivo import of this precursor, outer and inner membranes of yeast mitochondria become progressively “zippered” together, forming long stretches of close contact. Using this novel intermediate, the outer and inner mitochondrial membrane channels, which normally interact only transiently, can be tightly joined (both in vitro and in vivo), forming a stable association. This suggests a method for isolating the functional translocation complex as a single entity.

Phagocytosis of rod outer segments by retinal pigment epithelial cells requires αvβ5 integrin for binding but not for internalization

Phagocytosis of shed photoreceptor rod outer segments (ROS) by the retinal pigment epithelium (RPE) is essential for retinal function. Here, we demonstrate that this process requires αvβ5 integrin, rather than αvβ3 integrin utilized by systemic macrophages. Although adult rat RPE expressed both αvβ3 and αvβ5 integrins, only αvβ3 was expressed at birth, when the retina is immature and phagocytosis is absent. Expression of αvβ5 was first detected in RPE at PN7 and reached adult levels at PN11, just before onset of phagocytic activity. Interestingly, αvβ5 localized in vivo to the apical plasma membrane, facing the photoreceptors, and to intracellular vesicles, whereas αvβ3 was expressed basolaterally. Using quantitative fluorimaging to assess in vitro uptake of fluorescent particles by human (ARPE-19) and rat (RPE-J) cell lines, αvβ5 function-blocking antibodies were shown to reduce phagocytosis by drastically decreasing (85%) binding of ROS but not of latex beads. In agreement with a role for αvβ5 in phagocytosis, immunofluorescence experiments demonstrated codistribution of αvβ5 integrin with internalized ROS. Control experiments showed that blocking αvβ3 function with antibodies did not inhibit ROS phagocytosis and that αvβ3 did not colocalize with phagocytosed ROS. Taken together, our results indicate that the RPE requires the integrin receptor αvβ5 specifically for the binding of ROS and that phagocytosis involves internalization of a ROS-αvβ5 complex. αvβ5 integrin does not participate in phagocytosis by other phagocytic cells and is the first of the RPE receptors involved in ROS phagocytosis that may be specific for this process.

MCD4 Encodes a Conserved Endoplasmic Reticulum Membrane Protein Essential for Glycosylphosphatidylinositol Anchor Synthesis in Yeast

Glycosylphosphatidylinositol (GPI)-anchored proteins are cell surface-localized proteins that serve many important cellular functions. The pathway mediating synthesis and attachment of the GPI anchor to these proteins in eukaryotic cells is complex, highly conserved, and plays a critical role in the proper targeting, transport, and function of all GPI-anchored protein family members. In this article, we demonstrate that MCD4, an essential gene that was initially identified in a genetic screen to isolate Saccharomyces cerevisiae mutants defective for bud emergence, encodes a previously unidentified component of the GPI anchor synthesis pathway. Mcd4p is a multimembrane-spanning protein that localizes to the endoplasmic reticulum (ER) and contains a large NH2-terminal ER lumenal domain. We have also cloned the human MCD4 gene and found that Mcd4p is both highly conserved throughout eukaryotes and has two yeast homologues. Mcd4p’s lumenal domain contains three conserved motifs found in mammalian phosphodiesterases and nucleotide pyrophosphases; notably, the temperature-conditional MCD4 allele used for our studies (mcd4–174) harbors a single amino acid change in motif 2. The mcd4–174 mutant (1) is defective in ER-to-Golgi transport of GPI-anchored proteins (i.e., Gas1p) while other proteins (i.e., CPY) are unaffected; (2) secretes and releases (potentially up-regulated cell wall) proteins into the medium, suggesting a defect in cell wall integrity; and (3) exhibits marked morphological defects, most notably the accumulation of distorted, ER- and vesicle-like membranes. mcd4–174 cells synthesize all classes of inositolphosphoceramides, indicating that the GPI protein transport block is not due to deficient ceramide synthesis. However, mcd4–174 cells have a severe defect in incorporation of [3H]inositol into proteins and accumulate several previously uncharacterized [3H]inositol-labeled lipids whose properties are consistent with their being GPI precursors. Together, these studies demonstrate that MCD4 encodes a new, conserved component of the GPI anchor synthesis pathway and highlight the intimate connections between GPI anchoring, bud emergence, cell wall function, and feedback mechanisms likely to be involved in regulating each of these essential processes. A putative role for Mcd4p as participating in the modification of GPI anchors with side chain phosphoethanolamine is also discussed.

A family of genes required for maintenance of cell wall integrity and for the stress response in Saccharomyces cerevisiae

The PKC1–MPK1 pathway in yeast functions in the maintenance of cell wall integrity and in the stress response. We have identified a family of genes that are putative regulators of this pathway. WSC1, WSC2, and WSC3 encode predicted integral membrane proteins with a conserved cysteine motif and a WSC1–green fluorescence protein fusion protein localizes to the plasma membrane. Deletion of WSC results in phenotypes similar to mutants in the PKC1–MPK1 pathway and an increase in the activity of MPK1 upon a mild heat treatment is impaired in a wscΔ mutant. Genetic analysis places the function of WSC upstream of PKC1, suggesting that they play a role in its activation. We also find a genetic interaction between WSC and the RAS–cAMP pathway. The RAS–cAMP pathway is required for cell cycle progression and for the heat shock response. Overexpression of WSC suppresses the heat shock sensitivity of a strain in which RAS is hyperactivated and the heat shock sensitivity of a wscΔ strain is rescued by deletion of RAS2. The functional characteristics and cellular localization of WSC suggest that they may mediate intracellular responses to environmental stress in yeast.

Reciprocal electromechanical properties of rat prestin: The motor molecule from rat outer hair cells

Cochlear outer hair cells (OHCs) are responsible for the exquisite sensitivity, dynamic range, and frequency-resolving capacity of the mammalian hearing organ. These unique cells respond to an electrical stimulus with a cycle-by-cycle change in cell length that is mediated by molecular motors in the cells' basolateral membrane. Recent work identified prestin, a protein with similarity to pendrin-related anion transporters, as the OHC motor molecule. Here we show that heterologously expressed prestin from rat OHCs (rprestin) exhibits reciprocal electromechanical properties as known for the OHC motor protein. Upon electrical stimulation in the microchamber configuration, rprestin generates mechanical force with constant amplitude and phase up to a stimulus frequency of at least 20 kHz. Mechanical stimulation of rprestin in excised outside-out patches shifts the voltage dependence of the nonlinear capacitance characterizing the electrical properties of the molecule. The results indicate that rprestin is a molecular motor that displays reciprocal electromechanical properties over the entire frequency range relevant for mammalian hearing.

Red Bell Pepper Chromoplasts Exhibit in Vitro Import Competency and Membrane Targeting of Passenger Proteins from the Thylakoidal Sec and ΔpH Pathways but Not the Chloroplast Signal Recognition Particle Pathway1

Chloroplast to chromoplast development involves new synthesis and plastid localization of nuclear-encoded proteins, as well as changes in the organization of internal plastid membrane compartments. We have demonstrated that isolated red bell pepper (Capsicum annuum) chromoplasts contain the 75-kD component of the chloroplast outer envelope translocon (Toc75) and are capable of importing chloroplast precursors in an ATP-dependent fashion, indicating a functional general import apparatus. The isolated chromoplasts were able to further localize the 33- and 17-kD subunits of the photosystem II O2-evolution complex (OE33 and OE17, respectively), lumen-targeted precursors that utilize the thylakoidal Sec and ΔpH pathways, respectively, to the lumen of an internal membrane compartment. Chromoplasts contained the thylakoid Sec component protein, cpSecA, at levels comparable to chloroplasts. Routing of OE17 to the lumen was abolished by ionophores, suggesting that routing is dependent on a transmembrane ΔpH. The chloroplast signal recognition particle pathway precursor major photosystem II light-harvesting chlorophyll a/b protein failed to associate with chromoplast membranes and instead accumulated in the stroma following import. The Pftf (plastid fusion/translocation factor), a chromoplast protein, integrated into the internal membranes of chromoplasts during in vitro assays, and immunoblot analysis indicated that endogenous plastid fusion/translocation factor was also an integral membrane protein of chromoplasts. These data demonstrate that the internal membranes of chromoplasts are functional with respect to protein translocation on the thylakoid Sec and ΔpH pathways.

The spatial and temporal representation of a tone on the guinea pig basilar membrane

In the mammalian cochlea, the basilar membrane's (BM) mechanical responses are amplified, and frequency tuning is sharpened through active feedback from the electromotile outer hair cells (OHCs). To be effective, OHC feedback must be delivered to the correct region of the BM and introduced at the appropriate time in each cycle of BM displacement. To investigate when OHCs contribute to cochlear amplification, a laser-diode interferometer was used to measure tone-evoked BM displacements in the basal turn of the guinea pig cochlea. Measurements were made at multiple sites across the width of the BM, which are tuned to the same characteristic frequency (CF). In response to CF tones, the largest displacements occur in the OHC region and phase lead those measured beneath the outer pillar cells and adjacent to the spiral ligament by about 90°. Postmortem, responses beneath the OHCs are reduced by up to 65 dB, and all regions across the width of the BM move in unison. We suggest that OHCs amplify BM responses to CF tones when the BM is moving at maximum velocity. In regions of the BM where OHCs contribute to its motion, the responses are compressive and nonlinear. We measured the distribution of nonlinear compressive vibrations along the length of the BM in response to a single frequency tone and estimated that OHC amplification is restricted to a 1.25- to 1.40-mm length of BM centered on the CF place.

Resonant tectorial membrane motion in the inner ear: its crucial role in frequency tuning.

The tectorial membrane has long been postulated as playing a role in the exquisite sensitivity of the cochlea. In particular, it has been proposed that the tectorial membrane provides a second resonant system, in addition to that of the basilar membrane, which contributes to the amplification of the motion of the cochlear partition. Until now, technical difficulties had prevented vibration measurements of the tectorial membrane and, therefore, precluded direct evidence of a mechanical resonance. In the study reported here, the vibration of the tectorial membrane was measured in two orthogonal directions by using a novel method of combining laser interferometry with a photodiode technique. It is shown experimentally that the motion of the tectorial membrane is resonant at a frequency of 0.5 octave (oct) below the resonant frequency of the basilar membrane and polarized parallel to the reticular lamina. It is concluded that the resonant motion of the tectorial membrane is due to a parallel resonance between the mass of the tectorial membrane and the compliance of the stereocilia of the outer hair cells. Moreover, in combination with the contractile force of outer hair cells, it is proposed that inertial motion of the tectorial membrane provides the necessary conditions to allow positive feedback of mechanical energy into the cochlear partition, thereby amplifying and tuning the cochlear response.

Cloning and expression of a second photoreceptor-specific membrane retina guanylyl cyclase (RetGC), RetGC-2.

One of the membrane guanylyl cyclases (GCs), RetGC, is expressed predominantly in photoreceptors. No extracellular ligand has been described for RetGC, but it is sensitive to activation by a soluble 24-kDa protein (p24) and is inhibited by Ca2+. This enzyme is, therefore, thought to play a role in resynthesizing cGMP for photoreceptor recovery or adaptation. By screening a human retinal cDNA library at low stringency with the cytoplasmic domains from four cyclases, we cloned cDNAs encoding a membrane CG that is most closely related to RetGC. We have named this GC RetGC-2, and now term the initially described RetGC RetGC-1. By in situ hybridization, mRNA encoding RetGC-2 is found only in the outer nuclear layer and inner segments of photoreceptor cells. By using synthetic peptide antiserum specific for each RetGC subtype, RetGC-2 can be distinguished from RetGC-1 as a slightly smaller protein in immunoblots of bovine rod outer segments. Membrane GC activity of recombinant RetGC-2 expressed in human embryonic kidney 293 cells is stimulated by the activator p24 and is inhibited by Ca2+ with an EC50 value of 50-100 nM. Our data reveal a previously unappreciated diversity of photoreceptor GCs.

H-ras, K-ras, and inner plasma membrane raft proteins operate in nanoclusters with differential dependence on the actin cytoskeleton

Plasma membrane compartmentalization imposes lateral segregation on membrane proteins that is important for regulating signal transduction. We use computational modeling of immunogold spatial point patterns on intact plasma membrane sheets to test different models of inner plasma membrane organization. We find compartmentalization at the nanoscale level but show that a classical raft model of preexisting stable domains into which lipid raft proteins partition is incompatible with the spatial point patterns generated by the immunogold labeling of a palmitoylated raft marker protein. Rather, approximate to 30% of the raft protein exists in cholesterol-dependent nanoclusters, with approximate to 70% distributed as monomers. The cluster/monomer ratio (number of proteins in clusters/number of proteins outside clusters) is independent of expression level. H-rasG12V and K-rasG12V proteins also operate in nanoclusters with fixed cluster/monomer ratios that are independent of expression level. Detailed calibration of the immunogold imaging protocol suggests that radii of raft and RasG12V protein nanoclusters may be as small as 11 and 6 nm, respectively, and shows that the nanoclusters contain small numbers (6.0-7.7) of proteins. Raft nanoclusters do not form if the actin cytoskeleton is disassembled. The formation of K-rasG12V but not H-rasG12V nanoclusters also is actin-dependent. K-rasG12V but not H-rasG12V signaling is abrogated by actin cytoskeleton disassembly, which shows that nanoclustering is critical for Ras function. These findings argue against stable preexisting domains on the inner plasma membrane in favor of dynamic actively regulated nanoclusters similar to those proposed for the outer plasma membrane. RasG12V nanoclusters may facilitate the assembly of essential signal transduction complexes.

Microencapsulation strategies for islet transplantation

A variety of islet microencapsulation techniques have been investigated to establish which method provides the least occlusive barrier to net insulin release in vitro, and optimum biocompatibility for islet implantation in vivo. NMRI mouse islets have been microencapsulated with Na+ -alginate-poly-L-lysine (PLL)/poly-L-ornithine (PLO)-alginate, Ba2+ -alginate and agarose gels. Both free and microencapsulated islets responded to glucose challenge in static incubation and perifusion by significantly increasing their rate of insulin release and theophylline significantly potentiated the insulin response to glucose. While little insulin was released from microencapsulated islets after short term (2 hours) static incubation, significantly greater amounts were released in response to glucose challenge after extended (8-24 hours) incubation. However, insulin release from all types of microencapsulated islets was significantly reduced compared with free islets. Na+ -alginate-PLO-alginate microencapsulated islets were significantly more responsive to elevated glucose than Na+ -alginate-PLL-alginate microencapsulated islets, due to the enhanced porosity of PLO membranes. The outer alginate layer created a significant barrier to glucose/insulin exchange and reduced the insulin responsiveness of microencapsulated islets to glucose. Ba2+ -alginate membrane coated islets, generated by the density gradient method, were the most responsive to glucose challenge. Low concentrations of NG-monomethyl L-arginine (L-NMMA) had no significant effect on glucose stimulated insulin release from either free or microencapsulated islets. However, 1.0 mmol/1 L-NMMA significantly inhibited the insulin response of both free and microencapsulated islets to glucose challenge. In vivo work designed to evaluate the extent of pericapsular fibrosis after 28 days ip. and sc. implantation of microencapsulated islets into STZ-diabetic recipients, revealed that the inclusion of islets within microcapsules increased their immunogenicity and markedly increased the extent of pericapsular fibrosis. When the outer alginate layer was omitted from microcapsules, little or no pericapsular mononuclear cell deposition was observed. The subcutaneous site was not suitable for microencapsulated islet transplantation in NMRI recipient mice. Systemic immunosuppression using cyclosporin A was effective in preventing pericapsular mononuclear cell deposition, while L-NMMA loading into microcapsules had no significant effect on pericapsular fibrosis, although it did maintain the integrity of microencapsulated islets.

Development of a Composite ion-exchange membrane for PDMS-based microfluidic devices

The purpose of this research is to investigate potential methods to produce an ion-exchange membrane that can be integrated directly into a polydimethylsiloxane Lab-on-a-Chip or Micro-Total-Analysis-System. The majority of microfluidic membranes are based on creating microporous structures, because it allows flexibility in the choice of material such that it can match the material of the microfluidic chip. This cohesion between the material of the microfluidic chip and membrane is an important feature to prevent bonding difficulties which can lead to leaking and other practical problems. However, of the materials commonly used to manufacture microfluidic chips, there are none that provide the ion-exchange capability. The DuPont product Nafion{TM} is chosen as the ion-exchange membrane, a copolymer with high conductivity and selectivity to cations and suitable for many applications such as electrolysis of water and the chlor-alkali process. The use of such an ion-exchange membrane in microfluidics could have multiple advantages, but there is no reversible/irreversible bonding that occurs between PDMS and Nafion{TM}. In this project multiple methods of physical entrapment of the ion-exchange material inside a film of PDMS are attempted. Through the use of the inherent properties of PDMS, very inexpensive sugar granulate can be used to make an inexpensive membrane mould which does not interfere with the PDMS crosslinking process. After dissolving away this sacrificial mould material, Nafion{TM} is solidified in the irregular granulate holes. Nafion{TM} in this membrane is confined in the irregular shape of the PDMS openings. The outer structure of the membrane is all PDMS and can be attached easily and securely to any PDMS-based microfluidic device through reversible or irreversible PDMS/PDMS bonding. Through impedance measurement, the effectiveness of these integrated membranes are compared against plain Nafion{TM} films in simple sodium chloride solutions.

In-situ formation of solidified hydrogen thin-membrane targets using a pulse tube cryocooler

An account is given of the Central Laser Facility's work to produce a cryogenic hydrogen targetry system using a pulse tube cryocooler. Due to the increasing demand for low Z thin laser targets, CLF (in collaboration with TUD) have been developing a system which allows the production of solid hydrogen membranes by engineering a design which can achieve this remotely; enabling the gas injection, condensation and solidification of hydrogen without compromising the vacuum of the target chamber. A dynamic sealing mechanism was integrated which allows targets to be grown and then remotely exposed to open vacuum for laser interaction. Further research was conducted on the survivability of the cryogenic targets which concluded that a warm gas effect causes temperature spiking when exposing the solidified hydrogen to the outer vacuum. This effect was shown to be mitigated by improving the pumping capacity of the environment and reducing the minimum temperature obtainable on the target mount. This was achieved by developing a two-stage radiation shield encased with superinsulating blanketing; reducing the base temperature from 14 0.5 K to 7.2 0.2 K about the coldhead as well as improving temperature control stability following the installation of a high-performance temperature controller and sensor apparatus. The system was delivered experimentally and in July 2014 the first laser shots were taken upon hydrogen targets in the Vulcan TAP facility.