Publications by authors named "Atul N Parikh"

95 Publications

Recurrent dynamics of rupture transitions of giant lipid vesicles at solid surfaces.

Biophys J 2021 Feb 16;120(4):586-597. Epub 2021 Jan 16.

Department of Biomedical Engineering, University of California, Davis, California; Department of Chemistry, University of California, Davis, California; Department of Chemical Engineering, University of California, Davis, California; Department of Materials Science and Engineering, University of California, Davis, California. Electronic address:

Single giant unilamellar vesicles (GUVs) rupture spontaneously from their salt-laden suspension onto solid surfaces. At hydrophobic surfaces, the GUVs rupture via a recurrent, bouncing ball rhythm. During each contact, the GUVs, rendered tense by the substrate interactions, porate, and spread a molecularly transformed motif of a monomolecular layer on the hydrophobic surface from the point of contact in a symmetric manner. The competition from pore closure, however, limits the spreading and produces a daughter vesicle, which re-engages with the substrate. At solid hydrophilic surfaces, by contrast, GUVs rupture via a distinctly different recurrent burst-heal dynamics; during burst, single pores nucleate at the contact boundary of the adhering vesicles, facilitating asymmetric spreading and producing a "heart"-shaped membrane patch. During the healing phase, the competing pore closure produces a daughter vesicle. In both cases, the pattern of burst-reseal events repeats multiple times, splashing and spreading the vesicular fragments as bilayer patches at the solid surface in a pulsatory manner. These remarkable recurrent dynamics arise, not because of the elastic properties of the solid surface, but because the competition between membrane spreading and pore healing, prompted by the surface-energy-dependent adhesion, determine the course of the topological transition.
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http://dx.doi.org/10.1016/j.bpj.2021.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7896033PMC
February 2021

Interactions of different lipoproteins with supported phospholipid raft membrane (SPRM) patterns to understand similar in-vivo processes.

Biochim Biophys Acta Biomembr 2021 Mar 28;1863(3):183535. Epub 2020 Dec 28.

Department of Applied Science to Biomedical Engineering, University of California, Davis, CA 95616, United States of America.

To better understand how lipoproteins interact and enter endothelium and participate in cellular processes, we investigated preferential lipid partitioning of triglyceride rich lipoproteins (TGRL), chylomicrons (CM), low density lipoproteins (LDL), very low density lipoproteins (VLDL) and their lipolysis products using supported phospholipid raft membrane (SPRM) patterns. We prepared SPRM patterns with Texas red labeled phospholipid patterns and Marina blue labeled raft patterns and added Atto-520 labeled lipoproteins (TGRL, CM, VLDL, LDL) and their lipolysis products in separate experiments and characterized these interactions using fluorescence microscopy. We observed that VLDL and LDL preferentially interacted with raft patterns. In contrast the TGRL and lipolysed products of TGRL interacted with both the patterns, slightly elevated preference for raft patterns and CM and its lipolysis products showed greater affinity to phospholipid patterns. The clear preference of VLDL and LDL for raft patterns suggests that these lipoproteins associate with cholesterol and sphingomyelin rich lipid micro-domains during their early interactions with endothelial cells, leading to atherosclerosis.
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http://dx.doi.org/10.1016/j.bbamem.2020.183535DOI Listing
March 2021

Crystallization of Cholesterol in Phospholipid Membranes Follows Ostwald's Rule of Stages.

J Am Chem Soc 2020 12 21;142(52):21872-21882. Epub 2020 Dec 21.

School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, 639798 Singapore, Singapore.

Crystallization of membrane-embedded components within phospholipid bilayers represents a distinct class of phase transformation that occurs in structurally organized, molecularly crowded, and dimensionally constrained amphiphilic fluids. Using unstable supported lipid bilayers-transiently assembled via surface-mediated fusion and spreading of bicellar precursors containing supersaturating concentrations of cholesterol-we monitor here the morphological evolution and dynamics of cholesterol crystallization within the membrane media. We find that the three-dimensional (3D) crystallization of cholesterol from an unstable two-dimensional (2D) in-membrane state proceeds via well-defined sequence of intermediates, including filaments, rods, helices, and 2D rectangular plates, before transforming into three-dimensional quadrilateral crystals-characteristic triclinic habit of cholesterol monohydrate. Our observations thus demonstrate that these structurally distinct cholesterol polymorphs are related to one another, contrasting with the notion that they represent disparate crystal habits stabilized by differences in lipid environments. Moreover, these observations indicate that cholesterol crystallization within the membrane media follows nonclassical multistep crystallization governed by the heuristic "Ostwald's rule of stages", which predicts that the crystallization kinetics proceed down the free energy landscape in a multistage process where each successive phase transition incurs the smallest loss of free energy relative to its predecessor. Furthermore, we find that the well-known cholesterol extracting agent, β-cyclodextrin, acts by catalytically tipping the equilibrium in favor of crystal growth adding cholesterol from the membrane phase to the crystal in a layer-by-layer manner. Taken together, our results provide a new description of in-membrane cholesterol crystallization and may pave for a screening tool for identifying molecular candidates that target cholesterol crystals.
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http://dx.doi.org/10.1021/jacs.0c10674DOI Listing
December 2020

Coupled membrane lipid miscibility and phosphotyrosine-driven protein condensation phase transitions.

Biophys J 2021 Apr 24;120(7):1257-1265. Epub 2020 Sep 24.

Department of Chemistry, University of California, Berkeley, Berkeley, California; The Howard Hughes Medical Institute Summer Institute, Marine Biological Laboratory, Woods Hole, Massachusetts. Electronic address:

Lipid miscibility phase separation has long been considered to be a central element of cell membrane organization. More recently, protein condensation phase transitions, into three-dimensional droplets or in two-dimensional lattices on membrane surfaces, have emerged as another important organizational principle within cells. Here, we reconstitute the linker for activation of T cells (LAT):growth-factor-receptor-bound protein 2 (Grb2):son of sevenless (SOS) protein condensation on the surface of giant unilamellar vesicles capable of undergoing lipid phase separations. Our results indicate that the assembly of the protein condensate on the membrane surface can drive lipid phase separation. This phase transition occurs isothermally and is governed by tyrosine phosphorylation on LAT. Furthermore, we observe that the induced lipid phase separation drives localization of the SOS substrate, K-Ras, into the LAT:Grb2:SOS protein condensate.
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http://dx.doi.org/10.1016/j.bpj.2020.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8059084PMC
April 2021

Leaf Surface Topography Contributes to the Ability of on Leafy Greens to Resist Removal by Washing, Escape Disinfection With Chlorine, and Disperse Through Splash.

Front Microbiol 2020 17;11:1485. Epub 2020 Jul 17.

Department of Plant Pathology, University of California, Davis, Davis, CA, United States.

The attachment of foodborne pathogens to leaf surfaces is a complex process that involves multiple physical, chemical, and biological factors. Here, we report the results from a study designed to specifically determine the contribution of spinach leaf surface topography as it relates to leaf axis (abaxial and adaxial) and leaf age (15, 45, and 75 days old) to the ability of to resist removal by surface wash, to avoid inactivation by chlorine, and to disperse through splash impact. We used fresh spinach leaves, as well as so-called "replicasts" of spinach leaf surfaces in the elastomer polydimethylsiloxane to show that leaf vein density correlated positively with the failure to recover from surfaces, not only using a simple water wash and rinse, but also a more stringent wash protocol involving a detergent. Such failure was more pronounced when was surface-incubated at 24°C compared to 4°C, and in the presence, rather than absence, of nutrients. Leaf venation also contributed to the ability of to survive a 50 ppm available chlorine wash and to laterally disperse by splash impact. Our findings suggest that the topographical properties of the leafy green surface, which vary by leaf age and axis, may need to be taken into consideration when developing prevention or intervention strategies to enhance the microbial safety of leafy greens.
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http://dx.doi.org/10.3389/fmicb.2020.01485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7380079PMC
July 2020

Discovery and mechanistic characterization of a structurally-unique membrane active peptide.

Biochim Biophys Acta Biomembr 2020 10 18;1862(10):183394. Epub 2020 Jun 18.

Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, United States of America; Department of Chemistry, University of California, Davis, United States of America. Electronic address:

Membrane active peptides (MAPs) have gained wide interest due to their far reaching applications in drug discovery and drug delivery. The search for new MAPs, however, has been largely skewed with bias selecting for physicochemical parameters believed to be important for membrane activity, such as alpha helicity, cationicity and hydrophobicity. Here we carry out a search-and-find strategy to screen a 100,000-membered one-bead-one-compound (OBOC) combinatorial peptide library for lead compounds, agnostic of those physicochemical constraints. Such a synthetic strategy also permits expansion of our peptide repertoire to include unnatural amino acids. Using this approach, we discovered a structurally unique lead peptide LBF14, a linear 14-mer peptide, that induces gross morphological disruption of membranes, irrespective of membrane composition. Further, we demonstrate that the unique insertion mechanism of the peptide, visualized by spinning disc confocal microscopy and further analyzed by electron paramagnetic resonance measurements, may be the cause of this large scale membrane deformation. We also demonstrate the robustness, reproducibility, and potential application of this technique to discover and characterize new membrane active peptides that display activity by local insertion and subsequent allosteric effects leading to global membrane disruption.
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http://dx.doi.org/10.1016/j.bbamem.2020.183394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7478859PMC
October 2020

Response of microbial membranes to butanol: interdigitation vs. disorder.

Phys Chem Chem Phys 2019 Jun;21(22):11903-11915

Singapore Centre for Environmental Sciences Engineering (SCELSE), Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore.

Biobutanol production by fermentation is potentially a sustainable alternative to butanol production from fossil fuels. However, the toxicity of butanol to fermentative bacteria, resulting largely from cell membrane fluidization, limits production titers and is a major factor limiting the uptake of the technology. Here, studies were undertaken, in vitro and in silico, on the butanol effects on a representative bacterial (i.e. Escherichia coli) inner cell membrane. A critical butanol : lipid ratio for stability of 2 : 1 was observed, computationally, consistent with complete interdigitation. However, at this ratio the bilayer was ∼20% thicker than for full interdigitation. Furthermore, butanol intercalation induced acyl chain bending and increased disorder, measured as a 27% lateral diffusivity increase experimentally in a supported lipid bilayer. There was also a monophasic Tm reduction in butanol-treated large unilamellar vesicles. Both behaviours are inconsistent with an interdigitated gel. Butanol thus causes only partial interdigitation at physiological temperatures, due to butanol accumulating at the phospholipid headgroups. Acyl tail disordering (i.e. splaying and bending) fills the subsequent voids. Finally, butanol short-circuits the bilayer and creates a coupled system where interdigitated and splayed phospholipids coexist. These findings will inform the design of strategies targeting bilayer stability for increasing biobutanol production titers.
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http://dx.doi.org/10.1039/c9cp01469aDOI Listing
June 2019

A Chain-Elongated Oligophenylenevinylene Electrolyte Increases Microbial Membrane Stability.

Adv Mater 2019 May 25;31(18):e1808021. Epub 2019 Mar 25.

School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, 639798, Singapore.

A novel conjugated oligoelectrolyte (COE) material, named S6, is designed to have a lipid-bilayer stabilizing topology afforded by an extended oligophenylenevinylene backbone. S6 intercalates biological membranes acting as a hydrophobic support for glycerophospholipid acyl chains. Indeed, Escherichia coli treated with S6 exhibits a twofold improvement in butanol tolerance, a relevant feature to achieve within the general context of modifying microorganisms used in biofuel production. Filamentous growth, a morphological stress response to butanol toxicity in E. coli, is observed in untreated cells after incubation with 0.9% butanol (v/v), but is mitigated by S6 treatment. Real-time fluorescence imaging using giant unilamellar vesicles reveals the extent to which S6 counters membrane instability. Moreover, S6 also reduces butanol-induced lipopolysaccharide release from the outer membrane to further maintain cell integrity. These findings highlight a deliberate effort in the molecular design of a chain-elongated COE to stabilize microbial membranes against environmental challenges.
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http://dx.doi.org/10.1002/adma.201808021DOI Listing
May 2019

Minimal Reconstitution of Membranous Web Induced by a Vesicle-Peptide Sol-Gel Transition.

Biomacromolecules 2019 04 26;20(4):1709-1718. Epub 2019 Mar 26.

Centre for Biomimetic Sensor Science , Nanyang Technological University , 50 Nanyang Drive 637553 , Singapore.

Positive strand RNA viruses replicate in specialized niches called membranous web within the cytoplasm of host cells. These virus replication organelles sequester viral proteins, RNA, and a variety of host factors within a fluid, amorphous matrix of clusters of endoplasmic reticulum (ER) derived vesicles. They are thought to form by the actions of a nonstructural viral protein NS4B, which remodels the ER and produces dense lipid-protein condensates. Here, we used in vitro reconstitution to identify the minimal components and elucidate physical mechanisms driving the web formation. We found that the N-terminal amphipathic domain of NS4B (peptide 4BAH2) and phospholipid vesicles (∼100-200 nm in diameter) were sufficient to produce a gel-like, viscoelastic condensate. This condensate coexists with the surrounding aqueous phase and affords rapid exchange of molecules. Together, it recapitulates the essential properties of the virus-induced membranous web. Our data support a novel phase separation mechanism in which phospholipid vesicles provide a supramolecular template spatially organizing multiple self-associating peptides thereby generating programmable multivalency de novo and inducing macroscopic phase separation.
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http://dx.doi.org/10.1021/acs.biomac.9b00081DOI Listing
April 2019

Carbon Nanotube Porins in Amphiphilic Block Copolymers as Fully Synthetic Mimics of Biological Membranes.

Adv Mater 2018 Dec 17;30(51):e1803355. Epub 2018 Oct 17.

Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA, 94550, USA.

Biological membranes provide a fascinating example of a separation system that is multifunctional, tunable, precise, and efficient. Biomimetic membranes, which mimic the architecture of cellular membranes, have the potential to deliver significant improvements in specificity and permeability. Here, a fully synthetic biomimetic membrane is reported that incorporates ultra-efficient 1.5 nm diameter carbon nanotube porin (CNTPs) channels in a block-copolymer matrix. It is demonstrated that CNTPs maintain high proton and water permeability in these membranes. CNTPs can also mimic the behavior of biological gap junctions by forming bridges between vesicular compartments that allow transport of small molecules.
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http://dx.doi.org/10.1002/adma.201803355DOI Listing
December 2018

Permeability and Line-Tension-Dependent Response of Polyunsaturated Membranes to Osmotic Stresses.

Biophys J 2018 11 6;115(10):1942-1955. Epub 2018 Oct 6.

Departments of Biomedical Engineering, University of California, Davis, California; Chemistry, University of California, Davis, California; Chemical Engineering, University of California, Davis, California; Materials Science & Engineering, University of California, Davis, California. Electronic address:

The lipidome of plant plasma membranes-enriched in cellular phospholipids containing at least one polyunsaturated fatty acid tail and a variety of phytosterols and phytosphingolipids-is adapted to significant abiotic stresses. But how mesoscale membrane properties of these membranes such as permeability and deformability, which arise from their unique molecular compositions and corresponding lateral organization, facilitate response to global mechanical stresses is largely unknown. Here, using giant vesicles reconstituting mixtures of polyunsaturated lipids (soy phosphatidylcholine), glucosylceramide, and sitosterol common to plant membranes, we find that the membranes adopt "janus-like" domain morphologies and display anomalous solute permeabilities. The former textures the membrane with a single sterol-glucosylceramide-enriched, liquid-ordered domain separated from a liquid-disordered phase consisting primarily of soy phosphatidylcholine. When subject to osmotic downshifts, the giant unilamellar vesicles (GUVs) respond by transiently producing well-known swell-burst cycles. In each cycle, the influx of water swells the GUV, rendering the membrane tense. Subsequent rupture of the membrane through transient poration, which localizes in the liquid-disordered phase or at the domain boundaries, reduces the osmotic stress by expelling some of the excess osmolytes (and solvent) before sealing. When subject to abrupt hypertonic stress, they deform by nucleating buds at the domain phase boundaries. Remarkably, this incipient vesiculation is reversed in a statistically significant fraction of GUVs because of the interplay with solute permeation timescales, which render osmotic stresses short-lived. This, then, suggests a novel control mechanism in which an interplay of permeability and deformability regulates osmotically induced membrane deformation and limits vesiculation-induced loss of membrane material. Interestingly, recapitulation of such dynamic morphological reconfigurability-switching between budded and nonbudded morphologies-due to the interplay of membrane permeability, which temporally reverses the osmotic gradient, and domain boundaries, which select modes of deformations, might prove valuable in endowing synthetic cells with novel morphological responsiveness.
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http://dx.doi.org/10.1016/j.bpj.2018.09.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303272PMC
November 2018

Engineering the interface between lipid membranes and nanoporous gold: A study by quartz crystal microbalance with dissipation monitoring.

Biointerphases 2018 01 5;13(1):011002. Epub 2018 Jan 5.

Institute for Materials Research IMO, Hasselt University, Wetenschapspark 1, B-3590 Diepenbeek, Belgium and IMEC vzw, division IMOMEC, Wetenschapspark 1, B-3590 Diepenbeek, Belgium.

Nanoporous gold (np-Au) is a nanostructured metal with many desirable attributes. Despite the growing number of applications of nanoporous materials, there are still open questions regarding their fabrication and subsequent surface functionalization. For example, the hydrophobic nature of gold surfaces makes the formation of planar supported lipid layers challenging. Here, the authors engineer the interface between np-Au and 1,2-dioleoyl-sn-glycero-3-phosphocholine lipid layers using well-differentiated approaches based on vesicle adsorption and solvent exchange methods. The results reveal that the nanotopography of the np-Au surface plays a clear role in the vesicle adsorption process. Compared to vesicle adsorption, the solvent exchange method proves successful in the formation of planar supported lipid bilayers in both np-Au and planar Au surfaces, being less sensitive to the surface morphological features. The influence of nanostructured surfaces on lipid layer formation is determined by the driving mechanisms behind each process, i.e., the balance of adhesion and cohesion forces in vesicle adsorption and lyotropic lipid phase transitions in solvent exchange, respectively. A better understanding of such interactions will contribute to the development of a variety of applications, from electrochemical biosensors to drug screening and delivery systems, using nanoporous gold coated with stimuli-responsive lipid layers.
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http://dx.doi.org/10.1116/1.5010249DOI Listing
January 2018

Pulsatile Gating of Giant Vesicles Containing Macromolecular Crowding Agents Induced by Colligative Nonideality.

J Am Chem Soc 2018 01 5;140(2):691-699. Epub 2018 Jan 5.

Department of Mechanical and Aerospace Engineering, University of California San Diego , La Jolla, California 92093, United States.

The ability of large macromolecules to exhibit nontrivial deviations in colligative properties of their aqueous solutions is well-appreciated in polymer physics. Here, we show that this colligative nonideality subjects giant lipid vesicles containing inert macromolecular crowding agents to osmotic pressure differentials when bathed in small-molecule osmolytes at comparable concentrations. The ensuing influx of water across the semipermeable membrane induces characteristic swell-burst cycles: here, cyclical and damped oscillations in size, tension, and membrane phase separation occur en route to equilibration. Mediated by synchronized formation of transient pores, these cycles orchestrate pulsewise ejection of macromolecules from the vesicular interior reducing the osmotic differential in a stepwise manner. These experimental findings are fully corroborated by a theoretical model derived by explicitly incorporating the contributions of the solution viscosity, solute diffusivity, and the colligative nonideality of the osmotic pressure in a previously reported continuum description. Simulations based on this model account for the differences in the details of the noncolligatively induced swell-burst cycles, including numbers and periods of the repeating cycles, as well as pore lifetimes. Taken together, our observations recapitulate behaviors of vesicles and red blood cells experiencing sudden osmotic shocks due to large (hundreds of osmolars) differences in the concentrations of small molecule osmolytes and link intravesicular macromolecular crowding with membrane remodeling. They further suggest that any tendency for spontaneous overcrowding in single giant vesicles is opposed by osmotic stresses and requires independent specific interactions, such as associative chemical interactions or those between the crowders and the membrane boundary.
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http://dx.doi.org/10.1021/jacs.7b10192DOI Listing
January 2018

Pulsatile Lipid Vesicles under Osmotic Stress.

Biophys J 2017 Apr;112(8):1682-1691

Department of Mechanical and Aerospace Engineering, University of California San Diego, La Jolla, California. Electronic address:

The response of lipid bilayers to osmotic stress is an important part of cellular function. Recent experimental studies showed that when cell-sized giant unilamellar vesicles (GUVs) are exposed to hypotonic media, they respond to the osmotic assault by undergoing a cyclical sequence of swelling and bursting events, coupled to the membrane's compositional degrees of freedom. Here, we establish a fundamental and quantitative understanding of the essential pulsatile behavior of GUVs under hypotonic conditions by advancing a comprehensive theoretical model of vesicle dynamics. The model quantitatively captures the experimentally measured swell-burst parameters for single-component GUVs, and reveals that thermal fluctuations enable rate-dependent pore nucleation, driving the dynamics of the swell-burst cycles. We further extract constitutional scaling relationships between the pulsatile dynamics and GUV properties over multiple timescales. Our findings provide a fundamental framework that has the potential to guide future investigations on the nonequilibrium dynamics of vesicles under osmotic stress.
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http://dx.doi.org/10.1016/j.bpj.2017.03.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406380PMC
April 2017

HDL Glycoprotein Composition and Site-Specific Glycosylation Differentiates Between Clinical Groups and Affects IL-6 Secretion in Lipopolysaccharide-Stimulated Monocytes.

Sci Rep 2017 03 13;7:43728. Epub 2017 Mar 13.

Department of Nutrition, University of California, Davis, CA 95616, USA.

The goal of this pilot study was to determine whether HDL glycoprotein composition affects HDL's immunomodulatory function. HDL were purified from healthy controls (n = 13), subjects with metabolic syndrome (MetS) (n = 13), and diabetic hemodialysis (HD) patients (n = 24). Concentrations of HDL-bound serum amyloid A (SAA), lipopolysaccharide binding protein (LBP), apolipoprotein A-I (ApoA-I), apolipoprotein C-III (ApoC-III), α-1-antitrypsin (A1AT), and α-2-HS-glycoprotein (A2HSG); and the site-specific glycovariations of ApoC-III, A1AT, and A2HSG were measured. Secretion of interleukin 6 (IL-6) in lipopolysaccharide-stimulated monocytes was used as a prototypical assay of HDL's immunomodulatory capacity. HDL from HD patients were enriched in SAA, LBP, ApoC-III, di-sialylated ApoC-III (ApoC-III) and desialylated A2HSG. HDL that increased IL-6 secretion were enriched in ApoC-III, di-sialylated glycans at multiple A1AT glycosylation sites and desialylated A2HSG, and depleted in mono-sialylated ApoC-III (ApoC-III). Subgroup analysis on HD patients who experienced an infectious hospitalization event within 60 days (HD+) (n = 12), vs. those with no event (HD-) (n = 12) showed that HDL from HD+ patients were enriched in SAA but had lower levels of sialylation across glycoproteins. Our results demonstrate that HDL glycoprotein composition, including the site-specific glycosylation, differentiate between clinical groups, correlate with HDL's immunomodulatory capacity, and may be predictive of HDL's ability to protect from infection.
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http://dx.doi.org/10.1038/srep43728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347119PMC
March 2017

Spontaneous formation of nanometer scale tubular vesicles in aqueous mixtures of lipid and block copolymer amphiphiles.

Soft Matter 2017 Feb;13(6):1107-1115

Centre for Biomimetic Sensor Science, School of Materials Science & Engineering, Nanyang Technological University, Singapore 637553.

Many common amphiphiles self-assemble in water to produce heterogeneous populations of discrete and symmetric but polydisperse and multilamellar vesicles isolating the encapsulated aqueous core from the surrounding bulk. But when mixtures of amphiphiles of vastly different elastic properties co-assemble, their non-uniform molecular organization can stabilize lower symmetries and produce novel shapes. Here, using high resolution electron cryomicroscopy and tomography, we identify the spontaneous formation of a membrane morphology consisting of unilamellar tubular vesicles in dilute aqueous solutions of binary mixtures of two different amphiphiles of vastly different origins. Our results show that aqueous phase mixtures of a fluid-phase phospholipid and an amphiphilic block copolymer spontaneously assume a bimodal polymorphic character in a composition dependent manner: over a broad range of compositions (15-85 mol% polymer component), a tubular morphology co-exists with spherical vesicles. Strikingly, in the vicinity of equimolar compositions, an exclusively tubular morphology (L; diameter, ∼15 nm; length, >1 μm; core, ∼2.0 nm; wall, ∼5-6 nm) emerges in an apparent steady state. Theory suggests that the spontaneous stabilization of cylindrical vesicles, unaided by extraneous forces, requires a significant spontaneous bilayer curvature, which in turn necessitates a strongly asymmetric membrane composition. We confirm that such dramatic compositional asymmetry is indeed produced spontaneously in aqueous mixtures of a lipid and polymer through two independent biochemical assays - (1) reduction in the quenching of fluorophore-labeled lipids and (2) inhibition in the activity of externally added lipid-hydrolyzing phospholipase A2, resulting in a significant enrichment of the polymer component in the outer leaflet. Taken together, these results illustrate the coupling of the membrane shape with local composition through spontaneous curvature generation under conditions of asymmetric distribution of mixtures of disparate amphiphiles.
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http://dx.doi.org/10.1039/c6sm01753cDOI Listing
February 2017

Spontaneous Vesiculation and pH-Induced Disassembly of a Lysosomotropic Detergent: Impacts on Lysosomotropism and Lysosomal Delivery.

Langmuir 2016 12 12;32(50):13566-13575. Epub 2016 Dec 12.

Experimental Pathology, Department of Clinical and Experimental Medicine, Linköping, University , SE-581 85 Linköping, Sweden.

Lysosomotropic detergents (LDs) selectively rupture lysosomal membranes through mechanisms that have yet to be characterized. A consensus view, currently, holds that LDs, which are weakly basic, diffuse across cellular membranes as monomers in an uncharged state, and via protonation in the acidic lysosomal compartment, they become trapped, accumulate, and subsequently solubilize the membrane and induce lysosomal membrane permeabilization. Here we demonstrate that the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH) spontaneously assembles into vesicles at, and above, cytosolic pH, and that the vesicles disassemble as the pH reaches 6.4 or lower. The aggregation commences at concentrations below the range of those used in cell studies. Assembly and disassembly of the vesicles was studied via dynamic light scattering, zeta potential measurements, cryo-TEM, and fluorescence correlation spectroscopy and was found to be reversible via control of the pH. Aggregation of MSDH into closed vesicles under cytosolic conditions is at variance with the commonly held view of LD behavior, and we propose that endocytotic pathways should be considered as possible routes of LD entry into lysosomes. We further demonstrate that MSDH vesicles can be loaded with fluorophores via a solution transition from low to high pH, for subsequent release when the pH is lowered again. The ability to encapsulate molecular cargo into MSDH vesicles together with its ability to disaggregate at low pH and to permeabilize the lysosomal membrane presents an intriguing possibility to use MSDH as a delivery system.
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http://dx.doi.org/10.1021/acs.langmuir.6b03458DOI Listing
December 2016

Continuity of Monolayer-Bilayer Junctions for Localization of Lipid Raft Microdomains in Model Membranes.

Sci Rep 2016 05 27;6:26823. Epub 2016 May 27.

School of Electrical Engineering #032, Seoul National University, Kwanak P.O. Box 34, Seoul 151-600 Korea.

We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed between the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates.
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http://dx.doi.org/10.1038/srep26823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882513PMC
May 2016

Brownian Dynamics of Electrostatically Adhering Small Vesicles to a Membrane Surface Induces Domains and Probes Viscosity.

Langmuir 2016 05 17;32(21):5445-50. Epub 2016 May 17.

School of Materials Science and Engineering, Nanyang Technological University , 50 Nanyang Avenue 639798, Singapore.

Using single-particle tracking, we investigate the interaction of small unilamellar vesicles (SUVs) that are electrostatically tethered to the freestanding membrane of a giant unilamellar vesicle (GUV). We find that the surface mobility of the GUV-riding SUVs is Brownian, insensitive to the bulk viscosity, vesicle size, and vesicle fluidity but strongly altered by the viscosity of the underlying membrane. Analyzing the diffusional behavior of SUVs within the Saffman-Delbrück model for the dynamics of membrane inclusions supports the notion that the mobility of the small vesicles is coupled to that of dynamically induced lipid clusters within the target GUV membrane. The reversible binding also offers a nonperturbative means for measuring the viscosity of biomembranes, which is an important parameter in cell physiology and function.
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http://dx.doi.org/10.1021/acs.langmuir.6b00985DOI Listing
May 2016

Cholesterol Partition and Condensing Effect in Phase-Separated Ternary Mixture Lipid Multilayers.

Biophys J 2016 Mar;110(6):1355-66

Department of Physics, University of California-San Diego, La Jolla, California. Electronic address:

The cholesterol partitioning and condensing effect in the liquid-ordered (Lo) and liquid-disordered (Ld) phases were systematically investigated for ternary mixture lipid multilayers consisting of 1:1 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphocholine with varying concentrations of cholesterol. X-ray lamellar diffraction was used to deduce the electron density profiles of each phase. The cholesterol concentration in each phase was quantified by fitting of the electron density profiles with a newly invented basic lipid profile scaling method that minimizes the number of fitting parameters. The obtained cholesterol concentration in each phase versus total cholesterol concentration in the sample increases linearly for both phases. The condensing effect of cholesterol in ternary lipid mixtures was evaluated in terms of phosphate-to-phosphate distances, which together with the estimated cholesterol concentration in each phase was converted into an average area per molecule. In addition, the cholesterol position was determined to a precision of (±0.7Å) and an increase of disorder in the lipid packing in the Lo phase was observed for total cholesterol concentration of 20∼30%.
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http://dx.doi.org/10.1016/j.bpj.2016.02.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4816764PMC
March 2016

Mixing Water, Transducing Energy, and Shaping Membranes: Autonomously Self-Regulating Giant Vesicles.

Langmuir 2016 Mar 11;32(9):2151-63. Epub 2016 Feb 11.

Centre for Biomimetic Sensor Science, School of Materials Science & Engineering, Nanyang Technological University , Singapore 637553.

Giant lipid vesicles are topologically closed compartments bounded by semipermeable flexible shells, which isolate femto- to picoliter quantities of the aqueous core from the surrounding bulk. Although water equilibrates readily across vesicular walls (10(-2)-10(-3) cm(3) cm(-2) s(-1)), the passive permeation of solutes is strongly hindered. Furthermore, because of their large volume compressibility (∼10(9)-10(10) N m(-2)) and area expansion (10(2)-10(3) mN m(-1)) moduli, coupled with low bending rigidities (10(-19) N m), vesicular shells bend readily but resist volume compression and tolerate only a limited area expansion (∼5%). Consequently, vesicles experiencing solute concentration gradients dissipate the available chemical energy through the osmotic movement of water, producing dramatic shape transformations driven by surface-area-volume changes and sustained by the incompressibility of water and the flexible membrane interface. Upon immersion in a hypertonic bath, an increased surface-area-volume ratio promotes large-scale morphological remodeling, reducing symmetry and stabilizing unusual shapes determined, at equilibrium, by the minimal bending-energy configurations. By contrast, when subjected to a hypotonic bath, walls of giant vesicles lose their thermal undulation, accumulate mechanical tension, and, beyond a threshold swelling, exhibit remarkable oscillatory swell-burst cycles, with the latter characterized by damped, periodic oscillations in vesicle size, membrane tension, and phase behavior. This cyclical pattern of the osmotic influx of water, pressure, membrane tension, pore formation, and solute efflux suggests quasi-homeostatic self-regulatory behavior allowing vesicular compartments produced from simple molecular components, namely, water, osmolytes, and lipids, to sense and regulate their microenvironment in a negative feedback loop.
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http://dx.doi.org/10.1021/acs.langmuir.5b04470DOI Listing
March 2016

Cholesterol-Enriched Domain Formation Induced by Viral-Encoded, Membrane-Active Amphipathic Peptide.

Biophys J 2016 Jan;110(1):176-87

Biophysics Graduate Group, University of California, Davis, Davis, California; Department of Chemical Engineering & Materials Science, University of California, Davis, Davis, California; Centre for Biomimetic Sensor Science, Nanyang Technological University, Singapore; School of Materials Science and Engineering, Nanyang Technological University, Singapore; Department of Biomedical Engineering, University of California, Davis, Davis, California. Electronic address:

The α-helical (AH) domain of the hepatitis C virus nonstructural protein NS5A, anchored at the cytoplasmic leaflet of the endoplasmic reticulum, plays a role in viral replication. However, the peptides derived from this domain also exhibit remarkably broad-spectrum virocidal activity, raising questions about their modes of membrane association. Here, using giant lipid vesicles, we show that the AH peptide discriminates between membrane compositions. In cholesterol-containing membranes, peptide binding induces microdomain formation. By contrast, cholesterol-depleted membranes undergo global softening at elevated peptide concentrations. Furthermore, in mixed populations, the presence of ∼100 nm vesicles of viral dimensions suppresses these peptide-induced perturbations in giant unilamellar vesicles, suggesting size-dependent membrane association. These synergistic composition- and size-dependent interactions explain, in part, how the AH domain might on the one hand segregate molecules needed for viral assembly and on the other hand furnish peptides that exhibit broad-spectrum virocidal activity.
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http://dx.doi.org/10.1016/j.bpj.2015.11.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806215PMC
January 2016

Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

Sci Rep 2015 Nov 12;5:16432. Epub 2015 Nov 12.

Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, S-223 62 Lund, Sweden.

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.
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http://dx.doi.org/10.1038/srep16432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4642337PMC
November 2015

Medium Matters: Order through Fluctuations?

Authors:
Atul N Parikh

Biophys J 2015 Jun;108(12):2751-3

Departments of Biomedical Engineering and Chemical Engineering & Materials Science, University of California, Davis, Davis, California; Centre for Biomimetic Sensor Science, School of Materials Science & Engineering, Nanyang Technological University, Singapore. Electronic address:

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http://dx.doi.org/10.1016/j.bpj.2015.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472223PMC
June 2015

Lipid Membrane Deformation Accompanied by Disk-to-Ring Shape Transition of Cholesterol-Rich Domains.

J Am Chem Soc 2015 Jul 2;137(27):8692-5. Epub 2015 Jul 2.

§School of Electrical Engineering, Seoul National University, Seoul, Republic of Korea 151-742.

During vesicle budding or endocytosis, biomembranes undergo a series of lipid- and protein-mediated deformations involving cholesterol-enriched lipid rafts. If lipid rafts of high bending rigidities become confined to the incipient curved membrane topology such as a bud-neck interface, they can be expected to reform as ring-shaped rafts. Here, we report on the observation of a disk-to-ring shape morpho-chemical transition of a model membrane in the absence of geometric constraints. The raft shape transition is triggered by lateral compositional heterogeneity and is accompanied by membrane deformation in the vertical direction, which is detected by height-sensitive fluorescence interference contrast microscopy. Our results suggest that a flat membrane can become curved simply by dynamic changes in local chemical composition and shape transformation of cholesterol-rich domains.
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http://dx.doi.org/10.1021/jacs.5b04559DOI Listing
July 2015

Observation of Stripe Superstructure in the β-Two-Phase Coexistence Region of Cholesterol-Phospholipid Mixtures in Supported Membranes.

J Am Chem Soc 2014 Dec 26;136(49):16962-5. Epub 2014 Nov 26.

School of Materials Science and Engineering, ‡Centre for Biomimetic Sensor Science, and §School of Chemical and Biomedical Engineering, Nanyang Technological University , 50 Nanyang Avenue, Singapore 639798.

Visualization of phase coexistence in the β region of cholesterol-phospholipid mixtures consisting of high cholesterol concentrations has proved elusive in lipid bilayers. Here, using the solvent-assisted lipid bilayer approach to prepare supported membranes with high cholesterol fractions close to the cholesterol solubility limit, we report the observation of coexisting liquid phases using fluorescence microscopy. At ∼63 mol % cholesterol, supported membranes consisting of mixtures of DOPC and cholesterol exhibit large-area striping reminiscent of the stripe superstructures that characterize the proximity of the second critical point in the miscibility phase diagram. The properties of the two phases are consistent with condensed complex-rich and cholesterol-rich liquids. Both phases exhibit long-range lateral mobility, and diffusion through a given phase is favored over hopping across the phase boundary, producing an "archipelago effect" and a complex percolation path.
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http://dx.doi.org/10.1021/ja5082537DOI Listing
December 2014

Oscillatory phase separation in giant lipid vesicles induced by transmembrane osmotic differentials.

Elife 2014 Oct 15;3:e03695. Epub 2014 Oct 15.

School of Materials Science and Engineering, Nanyang Technological University, Nanyang, Singapore.

Giant lipid vesicles are closed compartments consisting of semi-permeable shells, which isolate femto- to pico-liter quantities of aqueous core from the bulk. Although water permeates readily across vesicular walls, passive permeation of solutes is hindered. In this study, we show that, when subject to a hypotonic bath, giant vesicles consisting of phase separating lipid mixtures undergo osmotic relaxation exhibiting damped oscillations in phase behavior, which is synchronized with swell-burst lytic cycles: in the swelled state, osmotic pressure and elevated membrane tension due to the influx of water promote domain formation. During bursting, solute leakage through transient pores relaxes the pressure and tension, replacing the domain texture by a uniform one. This isothermal phase transition--resulting from a well-coordinated sequence of mechanochemical events--suggests a complex emergent behavior allowing synthetic vesicles produced from simple components, namely, water, osmolytes, and lipids to sense and regulate their micro-environment.
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http://dx.doi.org/10.7554/eLife.03695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4197780PMC
October 2014

Formation of cholesterol-rich supported membranes using solvent-assisted lipid self-assembly.

Langmuir 2014 Nov 27;30(44):13345-52. Epub 2014 Oct 27.

School of Materials Science and Engineering, Nanyang Technological University , 50 Nanyang Avenue, Singapore 639798, Singapore.

This paper describes the application of a solvent-exchange method to prepare supported membranes containing high fractions of cholesterol (up to ∼57 mol %) in an apparent equilibrium. The method exploits the phenomenon of reverse-phase evaporation, in which the deposition of lipids in alcohol (e.g., isopropanol) is followed by the slow removal of the organic solvent from the water-alcohol mixture. This in turn induces a series of lyotropic phase transitions successively producing inverse-micelles, monomers, micelles, and vesicles in equilibrium with supported bilayers at the contacting solid surface. By using the standard cholesterol depletion by methyl-β-cyclodextrin treatment, a quartz crystal microbalance with dissipation monitoring assay confirms that the cholesterol concentration in the supported membranes is comparable to that in the surrounding bulk phase. A quantitative characterization of the biophysical properties of the resultant bilayer, including lateral diffusion constants and phase separation, using epifluorescence microscopy and atomic force microscopy establishes the formation of laterally contiguous supported lipid bilayers, which break into a characteristic domain-pattern of coexisting phases in a cholesterol concentration-dependent manner. With increasing cholesterol fraction in the supported bilayer, the size of the domains increases, ultimately yielding two-dimensional cholesterol bilayer domains near the solubility limit. A unique feature of the approach is that it enables preparation of supported membranes containing limiting concentrations of cholesterol near the solubility limit under equilibrium conditions, which cannot be obtained using conventional techniques (i.e., vesicle fusion).
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http://dx.doi.org/10.1021/la5034433DOI Listing
November 2014

Characterization of buried metal-molecule-metal junctions using Fourier transform infrared microspectroscopy.

Rev Sci Instrum 2014 Sep;85(9):094103

Department of Electrical and Computer Engineering, University of California, Davis, Davis, California 95616, USA.

We have devised an infrared spectromicroscopy based experimental configuration to enable structural characterization of buried molecular junctions. Our design utilizes a small mercury drop at the focal point of an infrared microscope to act as a mirror in studying metal-molecule-metal (MmM) junctions. An organic molecular monolayer is formed either directly on the mercury drop or on a thin, infrared (IR) semi-transparent layer of Au deposited onto an IR transparent, undoped silicon substrate. Following the formation of the monolayer, films on either metal can be examined independently using specular reflection spectroscopy. Furthermore, by bringing together the two monolayers, a buried molecular bilayer within the MmM junction can be characterized. Independent examination of each half of the junction prior to junction formation also allows probing any structural and/or conformational changes that occur as a result of forming the bilayer. Because our approach allows assembling and disassembling microscopic junctions by forming and withdrawing Hg drops onto the monolayer covered metal, spatial mapping of junctions can be performed simply by translating the location of the derivatized silicon wafer. Finally, the applicability of this technique for the longer-term studies of changes in molecular structure in the presence of electrical bias is discussed.
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http://dx.doi.org/10.1063/1.4896477DOI Listing
September 2014

Analysis of lipid phase behavior and protein conformational changes in nanolipoprotein particles upon entrapment in sol-gel-derived silica.

Langmuir 2014 Aug 6;30(32):9780-8. Epub 2014 Aug 6.

Department of Chemical Engineering and Materials Science and ‡Department of Biochemistry and Molecular Medicine, University of California Davis , Davis, California 95616, United States.

The entrapment of nanolipoprotein particles (NLPs) and liposomes in transparent, nanoporous silica gel derived from the precursor tetramethylorthosilicate was investigated. NLPs are discoidal patches of lipid bilayer that are belted by amphiphilic scaffold proteins and have an average thickness of 5 nm. The NLPs in this work had a diameter of roughly 15 nm and utilized membrane scaffold protein (MSP), a genetically altered variant of apolipoprotein A-I. Liposomes have previously been examined inside of silica sol-gels and have been shown to exhibit instability. This is attributed to their size (∼150 nm) and altered structure and constrained lipid dynamics upon entrapment within the nanometer-scale pores (5-50 nm) of the silica gel. By contrast, the dimensional match of NLPs with the intrinsic pore sizes of silica gel opens the possibility for their entrapment without disruption. Here we demonstrate that NLPs are more compatible with the nanometer-scale size of the porous environment by analysis of lipid phase behavior via fluorescence anisotropy and analysis of scaffold protein secondary structure via circular dichroism spectroscopy. Our results showed that the lipid phase behavior of NLPs entrapped inside of silica gel display closer resemblance to its solution behavior, more so than liposomes, and that the MSP in the NLPs maintain the high degree of α-helix secondary structure associated with functional protein-lipid interactions after entrapment. We also examined the effects of residual methanol on lipid phase behavior and the size of NLPs and found that it exerts different influences in solution and in silica gel; unlike in free solution, silica entrapment may be inhibiting NLP size increase and/or aggregation. These findings set precedence for a bioinorganic hybrid nanomaterial that could incorporate functional integral membrane proteins.
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http://dx.doi.org/10.1021/la5025058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140539PMC
August 2014