Ganglioside‐Selective Mechanosensitive Fluorescent Membrane Probes

The development of fluorescent probes to image forces in cells is an important challenge in chemistry and biology. Planarizable push‐pull probes have been introduced recently for this purpose. To provide most valuable information on forces in complex systems, these mechanosensitive ‘flipper’ probes will have to be localized by molecular recognition of targets of interest. Here we report fluorescent flippers that selectively recognize gangliosides on the surface of lipid bilayer membranes by formation of dynamic covalent boronate esters. The original flipper probes were equipped with 2‐fluorophenyl boronic acids and benzoboroxoles using consecutive triazole and oxime ligation. Evaluation was done in large unilamellar vesicles composed of EYPC/SM/CL/GM 40:40‐x:20:x to obtain mixed membranes with separate liquid‐disordered (L_d) and ganglioside (GM) containing liquid‐ordered (L_o) domains. With increasing GM concentration, fluorescence intensities increased and excitation maximum shifted to the red. Deconvolution of the spectra confirmed that these changes originate from a migration of the flipper probes from L_d to L_o domains upon binding to the gangliosides and thus the planarization in the more ordered environment. Control mechanophores without boronic acids failed to show the same response, and fructose partially inhibited the ganglioside sensitivity. These results demonstrate that it is possible to selectively accumulate mechanosensitive flipper probes in L_o domains and, more generally, that probe localization in complex membranes is possible and matters.     

Supported lipid bilayer formation by the giant vesicle fusion induced by vesicle–surface electrostatic attractive interaction

The effect of the electrostatic attractive force between giant unilamellar vesicles (GUVs) and the SiO₂ surface on the formation of a Ca²⁺-free supported lipid bilayer (SLB) was investigated by atomic force microscopy and fluorescence microscopy. When negatively charged GUVs were incubated for 1 h without Ca²⁺, the surface coverage of lipid bilayer was <1% on the SiO₂ surface. In contrast, a high coverage was obtained without addition of Ca²⁺ on the positively charged surface modified by aminopropyldimethylethoxysilane, and the coverage of SLBs decreased with increasing KCl concentrations. The thickness of the water layer under SLB was reduced by modification of APS.     

SOME RELATIONSHIPS BETWEEN ULTRAVIOLET LIGHT AND HEME-PROTEIN-INDUCED PEROXIDATIVE LIPID BREAKDOWN IN LIPOSOMES, AS REFLECTED BY FLUORESCENCE CHANGES: THE EFFECT OF NEGATIVE SURFACE CHARGE

Abstract— The water soluble, photolabile nitrene precursor,azidonaphthalene–2,7-disulfonic acid (ANDS) was encapsulated in small unilamellar, isoelectric (egg PC) or negatively charged (egg PC + dihexadecylphosphate) liposomes. The individual and combined effects of heme-proteins and UV irradiation on the fluorescence of these vesicles under aerobic conditions were studied. Consistent with the catalytic action of heme-proteins on lipid peroxidation and peroxide decomposition, addition of cytochrome c (positively charged) or catalase (negatively charged) to the vesicles elicited immediate formation of a fluorescence band at 470 nm, characteristic of Schiff bases that form from aldehyde byproducts of decomposing hydroperoxides. Ultraviolet irradiation of liposomes for 5 min caused no significant changes in the fluorescence spectrum, in spite of the radiolysis of ANDS inside the vesicles with consequent formation of nitrene radicals. When isoelectric vesicles were irradiated with UV light in the presence of cytochrome c or catalase, Schiff base formation was further increased by2–3 fold, which effect was not observed in the absence of internal ANDS, or in the presence of negative surface charge on the vesicles. These findings suggest that (a) UV irradiation, by itself, cannot trigger lipid decomposition even when it is assisted by photoproduced nitrene radicals, (b) there is a ternary synergism between UV light, heme-proteins and nitrene radicals in promoting peroxidative lipid breakdown, and (c) negative surface charge inhibits the above synergism, which effect is unlikely to be due to electrostatic interaction between the vesicles and the protein or the ANDS.     

Efficient formation of giant liposomes through the gentle hydration of phosphatidylcholine films doped with sugar

Giant liposomes, or giant vesicles, are cell-size (approximately 5-100 microm) compartments enclosed with phospholipid bilayers, and have often been used in biological research. They are usually generated using hydration methods, "electroformation" and "gentle hydration (or natural swelling)", in which dry lamellar films of phospholipids are hydrated with aqueous solutions. In gentle hydration, however, giant liposomes are difficult to prepare from an electrostatically neutral phospholipid because lipid lamellae cannot repel each other. In this study, we demonstrate the efficient formation of giant liposomes using the gentle hydration of neutral phospholipid (dioleoyl phosphatidylcholine, DOPC) dry films doped with nonelectrolytic monosaccharides (glucose, mannose, and fructose). A mixture of DOPC and such a sugar in an organic solvent (chloroform/methanol) was evaporated to form the films, which were then hydrated with distilled water or Tris buffers containing sodium chloride. Under these conditions, giant liposomes spontaneously formed rapidly and assumed a swollen cell-sized spherical shape with low lamellarity, whereas giant liposomes from pure DOPC films had multilamellar lipid layers, miscellaneous shapes and smaller sizes. This observation indicates that giant unilamellar vesicles (GUVs) of DOPC can be obtained efficiently through the gentle hydration of sugar-containing lipid dry films because repulsion between lipid lamellae is enhanced by the osmosis induced by dissolved sugar.     

Crystalline protein domains and lipid bilayer vesicle shape transformations

Cellular membranes can take on a variety of shapes to assist biological processes including endocytosis. Membrane-associated protein domains provide a possible mechanism for determining membrane curvature. We study the effect of tethered streptavidin protein crystals on the curvature of giant unilamellar vesicles (GUVs) using confocal, fluorescence, and differential interference contrast microscopy. Above a critical protein concentration, streptavidin domains align and percolate as they form, deforming GUVs into prolate spheroidal shapes in a size-dependent fashion. We propose a mechanism for this shape transformation based on domain growth and jamming. Osmotic deflation of streptavidin-coated GUVs reveals that the relatively rigid streptavidin protein domains resist membrane bending. Moreover, in contrast to highly curved protein domains that facilitate membrane budding, the relatively flat streptavidin domains prevent membrane budding under high osmotic stress. Thus, crystalline streptavidin domains are shown to have a stabilizing effect on lipid membranes. Our study gives insight into the mechanism for protein-mediated stabilization of cellular membranes.     

Fluorescence studies of the interactions of ubiquinol-10 with liposomes

Ubiquinone-10 plays a central role in energy production and its reduced form, ubiquinol-10 is also capable of acting as a potent radical scavenging antioxidant against membrane lipid peroxidation. Efficiency of this protection depends mostly on its localization in lipid bilayer. The intrinsic fluorescence of ubiquinol-10 and of the exogenous probe, Laurdan, has been used to determine the location of ubiquinol-10 in unilamellar liposomes of egg phosphatidylcholine (EggPC) and dimyristoyl phosphatidylcholine. Laurdan fluorescence moiety is positioned at the hydrophilic-hydrophobic interface of the phospholipid bilayer and its parameters reflect the membrane polarity and microheterogeneity, which we have used to explore the coexistence of microdomains with distinct physical properties. In liquid-crystalline bilayers ubiquinol has a short fluorescence lifetime (0.4 ns) and a high steady-state anisotropy. In a concentration-dependent manner, ubiquinol-10 influences the Laurdan excitation, emission and generalized polarization measurements. In EggPC liposomes ubiquinol-10 induces a decrease in membrane water mobility near the probe, while in dimyristoyl liposomes a decrease in the membrane water content was found. Moreover the presence of ubiquinol results in the formation of coexisting phospholipid domains of gel and liquid-crystalline phases. The results indicate that ubiquinol-10 molecules are mainly located at the polar-lipid interface, inducing changes in the physico-chemical properties of the bilayer microenvironment.     

Positioning lipid membrane domains in giant vesicles by micro-organization of aqueous cytoplasm mimic

We report localization of lipid membrane microdomains to specific "poles" of asymmetric giant vesicles (GVs) in response to local internal composition. Interior aqueous microdomains were generated in a simple model cytoplasm composed of a poly(ethyleneglycol) (PEG)/dextran aqueous two-phase system (ATPS) encapsulated in the vesicles. The GV membrane composition used here was a modification of a DOPC/DPPC/cholesterol mixture known to form micrometer-scale liquid ordered and liquid disordered domains; we added lipids with PEG 2000 Da-modified headgroups. Osmotically induced budding of the ATPS-containing GVs led to structures where the PEG-rich and dextran-rich interior aqueous phases were in contact with different regions of the vesicle membrane. Liquid ordered (L o) membrane domains rich in PEG-terminated lipids preferentially coated the PEG-rich aqueous phase vesicle "body", while coexisting liquid disordered (L d) membrane domains coated the dextran-rich aqueous phase "bud". Membrane domain positioning resulted from interactions between lipid headgroups and the interior aqueous polymer solutions, e.g., PEGylated headgroups with PEG and dextran polymers. Heating resulted first in patchy membranes where L o and L d domains no longer showed any preference for coating the PEG-rich vs dextran-rich interior aqueous volumes, and eventually complete lipid mixing. Upon cooling lipid domains again coated their preferred interior aqueous microvolume. This work shows that nonspecific interactions between interior aqueous contents and the membrane that encapsulates them can drive local chemical heterogeneity, and offers a primitive experimental model for membrane and cytoplasmic polarity in biological cells.     

巨型囊泡的侧向相分离与出芽

用电形成法制备含三组分二油酰磷脂酰胆碱(DOPC)/二棕榈酰磷脂酰胆碱(DPPC)/胆固醇(Chol)的巨型囊泡, 以TR-DPPE为荧光染色剂, 在荧光显微镜下直接观察膜的侧向相分离与微区相凸起出芽的耦合. 发现囊泡膜内的相分离具有诱导期, 相分离速度很快, 形成的微相区在整个囊泡球面上均衡分布. 各微相区的出芽不是同时进行, 为逐个随机发生. 每次出芽的时间小于0.5 s. 分相与出芽的耦合使球面上的不同微区之间不会相互融合成更大的微区.     

Membrane domain-disrupting effects of 4-substitued cholesterol derivatives

A wide range of cellular functions are thought to be regulated not only by the activity of membrane proteins, but also by the local membrane organization, including domains of specific lipid composition. Thus, molecules and drugs targeting and disrupting this lipid pattern, particularly of the plasma membrane, will not only help to investigate the role of membrane domains in cell biology, but might also be interesting candidates for therapy. We have identified three 4-substituted cholesterol derivatives that are able to induce a domain-disrupting effect in model membranes. When applied to giant unilamellar vesicles displaying liquid-ordered-liquid-disordered phase coexistence, extensive reorganization of the membrane can be observed, such as the budding of membrane tubules or changes in the geometry of the domains, to the point of complete abolition of phase separation. In this case, the resulting membranes display a fluidity intermediate between those of liquid-disordered and liquid-ordered phases.     

Formation of tubules and giant vesicles from large multilamellar vesicles

This study reports an observation of submicrometer multilamellar vesicles (MLVs) prepared by simply freeze-thawing a phospholipid dispersion at full hydration that transformed into giant vesicles (GVs) and tubules (TUs) when confined between microscope glass slides. Cover slide cleaning and surface treatment did not hamper the formation of GVs or TUs. However, when small unilamellar vesicles (SUV) were prepared or when MLVs were not confined but rather freely moved between the glass slides or when the phospholipid was in its gel phase, neither GVs nor TUs were observed. Altogether, our results suggested that MLVs would play a role as a lipid reservoir and that the liquid flow between the glass slides induces the peeling of the external bilayers, yielding the formation of tubules and giant unilamellar vesicles.     

聚醚氨酯的微区形态

<正> 聚醚氨酯热塑弹性体是属于(AB)n类型的线型多嵌段共聚物,包括交替的硬段和软段单元.自从1966年Cooper和Tobolsky首先提出聚氨酯具有微相分离的本体结构之假设以后,至今已有大量文献报道了这类材料结构形态与性能关系的研究结果. Wilkes和Koberstein等使用SAXS研究了聚氨酯体系的形态特征.一般认为,聚氨酯材料的微相结构包括一个叠层状或类似叠层状形态,由相分离的软段和硬段组成,平均尺寸为100A的数量级,在软硬段微区之间还存在一相混合的过渡区,其厚度可以从几个埃至几十个埃.然而,SAXS虽然能够高分辨地给出多相体系相分离情况的定     

DNA-Based Artificial Receptors as Transmembrane Signal Transduction Systems for Protocellular Communication

The ability to reproduce signal transduction and cellular communication in artificial cell systems is significant in synthetic protobiology. Here, we describe an artificial transmembrane signal transduction through low pH-mediated formation of the i-motif and dimerization of DNA-based artificial membrane receptors, which is coupled to the occurrence of fluorescence resonance energy transfer and the activation of G-quadruplex/hemin-mediated fluorescence amplification inside giant unilamellar vesicles. Moreover, an intercellular signal communication model is established when the extravesicular H⁺ input is replaced by coacervate microdroplets, which activate the dimerization of the artificial receptors, and subsequent fluorescence production or polymerization in giant unilamellar vesicles. This study represents a crucial step towards designing artificial signalling systems with environmental response, and provides an opportunity to establish signalling networks in protocell colonies.     

Langmuir-Blodgett膜超分子SM/DOPC/Chol三元脂系的原子力显微镜观察

用原子力显微镜研究了胆固醇(Chol)对鞘磷脂(SM)/1,2-二油酸甘油-3-磷脂酰胆碱(DOPC)二元脂系统结构的影响和神经酰胺对SM/DOPC/Chol三元脂系统结构的影响. 实验发现, 在SM/DOPC二元脂系统中, 胆固醇和带饱和脂肪酸链的磷脂发生相互作用形成微区结构, 随着胆固醇含量的增加, 微区的面积逐渐增大, 形成了稳定的片层结构. 当把神经酰胺加入到等摩尔配比的SM/DOPC/Chol三元脂系统中时, 随着神经酰胺比例的增加, 先形成紧密的聚集态结构, 然后逐渐演变成具有特定微区的网状结构. 研究结果表明, 微区的形成主要是由分子不同的官能团之间的相互作用所决定, 这可能在细胞信号传导等生理活动中起到重要的作用.     

Visualization and Quantification of Transmembrane Ion Transport into Giant Unilamellar Vesicles

Transmembrane ion transporters (ionophores) are widely investigated as supramolecular agents with potential for biological activity. Tests are usually performed in synthetic membranes that are assembled into large unilamellar vesicles (LUVs). However transport must be followed through bulk properties of the vesicle suspension, because LUVs are too small for individual study. An alternative approach is described whereby ion transport can be revealed and quantified through direct observation. The method employs giant unilamellar vesicles (GUVs), which are 20–60 μm in diameter and readily imaged by light microscopy. This allows characterization of individual GUVs containing transporter molecules, followed by studies of transport through fluorescence emission from encapsulated indicators. The method provides new levels of certainty and relevance, given that the GUVs are similar in size to living cells. It has been demonstrated using a highly active anion carrier, and should aid the development of compounds for treating channelopathies such as cystic fibrosis.     

Fluid supported lipid bilayers containing monosialoganglioside GM1: a QCM-D and FRAP study

In an effort to use model fluid membranes for immunological studies, we compared the formation of planar phospholipid bilayers supported on silicon dioxide surfaces with and without incorporation of glycolipids as the antigen for in situ antibody binding. Dynamic light scattering measurements did not differentiate the hydrodynamic volumes of extruded small unilamellar vesicles (E-SUVs) containing physiologically relevant concentrations (0.5-5 mol%) of monosialoganglioside GM1 (GM1) from exclusive egg yolk L-alpha-phosphatidylcholine (egg PC) E-SUVs. However, quantifiable differences in deposition mass and dissipative energy loss emerged in the transformation of 5 mol% GM1/95 mol% egg PC E-SUVs to planar supported lipid bilayers (PSLBs) by vesicle fusion on thermally evaporated SiO2, as monitored by the quartz crystal microbalance with dissipation (QCM-D) technique. Compared to the 100 mol% egg PC bilayers on the same surface, E-SUVs containing 5 mol% GM1 reached a approximately 12% higher mass and a lower dissipative energy loss during bilayer transformation. PSLBs with 5 mol% GM1 are approximately 18% heavier than 100 mol% egg PC and approximately 11% smaller in projected area per lipid, indicating an increased rigidity and a tighter packing. Subsequent binding of polyclonal immunoglobulin G anti-GM1 to the PSLBs was performed in situ and showed specificity. The anti-GM1 to GM1 ratios at equilibrium were roughly proportional to the concentrations of anti-GM1 administered in the solution. Fluorescence recovery after photobleaching was utilized to verify the retained, albeit reduced lateral fluidity of the supported membranes. Five moles percentage of GM1 membranes (GM1 to PC ratio approximately 1:19) decorated with 1 mol% N-(Texas Red sulfonyl)-1,2-dihexadecanoyl-sn-glycerol-3-phosphoethanolamine (Texas Red DHPE) exhibited an approximately 16% lower diffusion coefficient of 1.32+/-0.06 microm2/s, compared to 1.58+/-0.04 microm2/s for egg PC membranes without GM1 (p<0.01). The changes in vesicle properties and membrane lateral fluidity are attributed to the interactions of GM1 with itself and GM1 with other membrane lipids. This system allows for molecules of interest such as GM1 to exist on a more biologically relevant surface than those used in conventional methods such as ELISA. Our analysis of rabbit serum antibodies binding to GM1 demonstrates this platform can be used to test for the presence of anti-lipid antibodies in serum.     

Partitioning of dual-lipidated peptides into membrane microdomains: lipid sorting vs peptide aggregation

The lateral membrane organization and phase behavior of the lipid mixture DMPC(di-C(14))/DSPC(di-C(18))/cholesterol (0-33 mol %) with and without an incorporated fluorescence-labeled palmitoyl/farnesyl dual-lipidated peptide, BODIPY-Gly-Cys(Pal)-Met-Gly-Leu-Pro-Cys(Far)-OMe, which represents a membrane recognition model system for Ras proteins, was studied by two-photon excitation fluorescence microscopy. Measurements were performed on giant unilamellar vesicles (GUVs) over a large temperature range, ranging from 30 to 80 degrees C to cover different lipid phase states (all-gel, fluid/gel, liquid-ordered, all-fluid). At temperatures where the fluid-gel coexistence region of the pure binary phospholipid system occurs, large-scale concentration fluctuations appear. Incorporation of cholesterol levels up to 33 mol % leads to a significant increase of conformational order in the membrane system and a reduction of large domain structures. Adding the peptide leads to dramatic changes in the lateral organization of the membrane. With cholesterol present, a phase separation is induced by a lipid sorting mechanism owing to the high affinity of the lipidated peptide to a fluid, DMPC-rich environment. This phase separation leads to the formation of peptide-containing domains with high fluorescence intensity that become progressively smaller with decreasing temperature. As a result, the local concentration of the peptide increases steadily within the confines of the shrinking domains. At the lowest temperatures, where the acyl-chain order parameter of the membrane has already drastically increased and the membrane achieves a liquid-ordered character, an efficient lipid sorting mechanism is no longer supported and aggregation of the peptide into small clusters prevails. We can conclude that palmitoyl/farnesyl dual-lipidated peptides do not associate with liquid-ordered or gel-like domains in phase-separated bilayer membranes. In particular, the study shows the interesting ability of the peptide to induce formation of fluid microdomains at physiologically relevant cholesterol concentrations, and this effect very much depends on the concentration of fluid vs ordered lipid molecules.     

Hypelcin A,an α-aminoisobutyric acid containing antibiotic peptide,induced fusion of egg yolk-l-α-phosphatidylcholine small unilamellar vesicles

Hypelcin A, an α-aminoisobutyric acid-containing antibiotic peptide inducing fusion of egg yolk-l-α-phosphatidylcholine (egg PC) small unilamellar vesicles (SUVs), was investigated by lipid-mixing assay based on resonanceenergy transfer between fluorescent probes, electron microscopy, light scattering, and¹H-nuclear magnetic-resonance spectroscopy. At a high peptide-to-lipid ratio of approximately 1:5, the peptide fuses several SUVs of 20–30 nm in diameter into a 40–100 nm vesicle. Under mild conditions where the permeability enhancement (leakage of a trapped fluorescent dye, calcein) of lipid bilayers are observed (peptide to lipid ratios around 1/100), the fusion of the SUVs also occurs, although the fusion requires a somewhat larger amount of the peptide than the leakage does. Furthermore, at higher lipid concentrations, where the aggregation step is sufficiently rapid, the fusion rate is determined by the amount of the membrane bound peptide per lipid molecule, as is the leakage rate. In contrast, for egg PC large unilamellar vesicles (110 nm), hypelcin A induces the leakage, but not the fusion. We conclude that the leakage is not due to the fusion.     

Visualizing association of N-ras in lipid microdomains: influence of domain structure and interfacial adsorption

In this study, two-photon fluorescence microscopy on giant unilamellar vesicles and tapping-mode atomic force microscopy (AFM) are applied to follow the insertion of a fluorescently (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, BODIPY) labeled and completely lipidated (hexadecylated and farnesylated) N-Ras protein into heterogeneous lipid bilayer systems. The bilayers consist of the canonical raft mixture 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), sphingomyelin, and cholesterol, which-depending on the concentration of the constituents-separates into liquid-disordered (l(d)), liquid-ordered (l(o)), and solid-ordered (s(o)) phases. The results provide direct evidence that partitioning of N-Ras occurs preferentially into liquid-disordered lipid domains, which is also reflected in a faster kinetics of incorporation into the fluid lipid bilayers. The phase sequence of preferential binding of N-Ras to mixed-domain lipid vesicles is l(d) > l(o) > s(o). Intriguingly, we detect, using the better spatial resolution of AFM, also a large proportion of the lipidated protein located at the l(d)/l(o) phase boundary, thus leading to a favorable decrease in line tension that is associated with the rim of the demixed phases. Such an interfacial adsorption effect may serve as an alternative vehicle for association processes of signaling proteins in membranes.     

Phase behavior and the partitioning of caveolin-1 scaffolding domain peptides in model lipid bilayers

The membrane binding and model lipid raft interaction of synthetic peptides derived from the caveolin scaffolding domain (CSD) of the protein caveolin-1 have been investigated. CSD peptides bind preferentially to liquid-disordered domains in model lipid bilayers composed of cholesterol and an equimolar ratio of dioleoylphosphatidylcholine (DOPC) and brain sphingomyelin. Three caveolin-1 peptides were studied: the scaffolding domain (residues 83-101), a water-insoluble construct containing residues 89-101, and a water-soluble construct containing residues 89-101. Confocal and fluorescence microscopy investigation shows that the caveolin-1 peptides bind to the more fluid cholesterol-poor phase. The binding of the water-soluble peptide to lipid bilayers was measured using fluorescence correlation spectroscopy (FCS). We measured molar partition coefficients of 10(4) M(-1) between the soluble peptide and phase-separated lipid bilayers and 10(3) M(-1) between the soluble peptide and bilayers with a single liquid phase. Partial phase diagrams for our phase-separating lipid mixture with added caveolin-1 peptides were measured using fluorescence microscopy. The water-soluble peptide did not change the phase morphology or the miscibility transition in giant unilamellar vesicles (GUVs); however, the water-insoluble and full-length CSD peptides lowered the liquid-liquid melting temperature.     

Structural effects of a basic peptide on the organization of dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylserine membranes: a fluorescent resonance energy transfer study

We studied the effect of a model basic peptide, hexalysiltryptophan, on the organization of dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylserine unilamellar vesicles by means of fluorescent resonance energy transfer (FRET) between fluorescently labeled phospholipids. Several FRET theoretical models assuming different bilayer geometries and probe distributions were fitted to the time-resolved data. The experiments were carried out at two temperatures in different regions of the lipid mixture phase diagram. At 45 degrees C, the expected gel/fluid phase separation was verified by model fitting in peptide-free vesicles, which from the FRET approach means that domains are larger than approximately 200 A. No noticeable alteration of membrane organization was detected upon increasing the peptide concentration. At variance, for the single fluid phase at 60 degrees C, there was a large increase in FRET efficiency upon peptide addition to the lipid vesicles, mainly caused by peptide-induced vesicle aggregation. The system gradually changed from unilamellar lipid vesicles to a multibilayer geometry, and a limit lamellar repeat distance of approximately 57 A was recovered. Furthermore, no evidence for lateral domain formation on the FRET length scale was found at this temperature, the cationic peptide being only able to induce local lipid demixing, causing a short-range sequestration of 2-3 acidic lipids around each surface-adsorbed peptide.