Lipid Transition Research Articles

Uniaxial Symmetry Breaking in Bacteriorhodopsin at the Thermal Phase Transition of Lipids of Purple Membranes.

The article correlates between symmetry breaking and phase transition. An analogy, extending from physics to biology, is known to exist between these two topics. Bacteriorhodopsin (bR) as a paradigm of membrane proteins has been used as a case study in the present work. The bR, as the sole protein embedded in what is called a purple membrane (PM), has attracted widespread interest in bionanotechnological applications. The lipids of PM have a crucial role in maintaining the crystal lattice of bR inside PM. For this reason, the present work has been concerned with elucidating the thermal phase transition properties of the PM lipids in orthogonal directions. The results indicated that the axial symmetry of bR exhibits considerable changes occurring at the thermal phase transition of lipids. These changes are brought by an anomaly observed in the time course of orthogonal electric responses during the application of thermal fields on PM. The observed anomaly may bear on symmetry breaking in bR occurring at the phase transition of lipids based on such analogy found between symmetry breaking and phase transition. Lipid-protein interactions may underlie the broken axial symmetry of bR at such lipid thermal transition of PM. Accordingly, thermally perturbed axial symmetry of bR may be of biological relevance relying on the essence of the crystal lattice of bR. Most importantly, a question has to be raised in the present study: Can bR, as a helical protein with broken axial symmetry, affect the symmetry breaking of helical light? This may be of potential technical applications based on a recent discovery that bR breaks the symmetry of helical light.

Interactions of Galloylated Polyphenols with a Simple Gram-Negative Bacterial Membrane Lipid Model.

Differential scanning calorimetry (DSC) was used to explore the interactions of isolated polyphenolic compounds, including (-)-epigallocatechin gallate ((-)-EGCg), tellimagrandins I and II (Tel-I and Tel-II), and 1,2,3,4,6-penta-O-galloyl-d-glucose (PGG), with a model Gram-negative bacterial membrane with a view to investigating their antimicrobial properties. The model membranes comprised 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), fabricated to mimic the domain formation observed in natural membranes, as well as ideally mixed lipid vesicles for the interaction with (-)-EGCg. Polyphenols induced changes in lipid mixing/de-mixing depending on the method of vesicle preparation, as was clearly evidenced by alterations in the lipid transition temperatures. There was a distinct affinity of the polyphenols for the DPPG lipid component, which was attributed to the electrostatic interactions between the polyphenolic galloyl moieties and the lipid headgroups. These interactions were found to operate through either the stabilization of the lipid headgroups by the polyphenols or the insertion of the polyphenols into the membrane itself. Structural attributes of the polyphenols, including the number of galloyl groups, the hydrophobicity quantified by partition coefficients (logP), and structural flexibility, exhibited a correlation with the temperature transitions observed in the DSC measurements. This study furthers our understanding of the intricate interplay between the structural features of polyphenolic compounds and their interactions with model bacterial membrane vesicles towards the exploitation of polyphenols as antimicrobials.

Skin Barrier Fine Tuning through Low-Temperature Lipid Chain Transition

The lipids in the mammalian stratum corneum (SC) adopt an unusually rigid arrangement to form a vital barrier preventing water loss and harmful environmental impacts. Just above the physiological temperature, a subset of barrier lipids undergoes a phase transition from a very tight orthorhombic to a looser hexagonal arrangement and vice versa. The purpose of this lipid transition in skin physiology is unknown. Permeability experiments on isolated human SC indicated that the transition affects the activation energy for a model compound that prefers lateral movement along lipid layers but not for water or a large polymer that would cross the SC through the pore pathway. The orthorhombic phase content of SC lipids, as determined by infrared spectroscopy, was also modulated by (de)hydration. Spontaneous rearrangement of human SC lipid monolayers into 10 nm higher multilamellar islets at 32-37 °C but not at room temperature was revealed by atomic force microscopy. Our findings add to our knowledge of fundamental skin physiology suggesting a fine temperature- and hydration-controlled switch from fluid lipids (required for lipid barrier assembly) to rigid and tightly packed lipids in the mature SC (necessary for the water and permeability barriers).

Hypo-Osmotic Stress and Pore-Forming Toxins Adjust the Lipid Order in Sheep Red Blood Cell Membranes.

Lipid ordering in cell membranes has been increasingly recognized as an important factor in establishing and regulating a large variety of biological functions. Multiple investigations into lipid organization focused on assessing ordering from temperature-induced phase transitions, which are often well outside the physiological range. However, particular stresses elicited by environmental factors, such as hypo-osmotic stress or protein insertion into membranes, with respect to changes in lipid status and ordering at constant temperature are insufficiently described. To fill these gaps in our knowledge, we exploited the well-established ability of environmentally sensitive membrane probes to detect intramembrane changes at the molecular level. Our steady state fluorescence spectroscopy experiments focused on assessing changes in optical responses of Laurdan and diphenylhexatriene upon exposure of red blood cells to hypo-osmotic stress and pore-forming toxins at room temperature. We verified our utilized experimental systems by a direct comparison of the results with prior reports on artificial membranes and cholesterol-depleted membranes undergoing temperature changes. The significant changes observed in the lipid order after exposure to hypo-osmotic stress or pore-forming toxins resembled phase transitions of lipids in membranes, which we explained by considering the short-range interactions between membrane components and the hydrophobic mismatch between membrane thickness and inserted proteins. Our results suggest that measurements of optical responses from the membrane probes constitute an appropriate method for assessing the status of lipids and phase transitions in target membranes exposed to mechanical stresses or upon the insertion of transmembrane proteins.

The ultrastructural function of MLN64 in the late endosome–mitochondria membrane contact sites in placental cells

The close apposition between two different organelles is critical in essential processes such as ion homeostasis, signaling, and lipid transition. However, information related to the structural features of membrane contact sites (MCSs) is limited. This study used immuno-electron microscopy and immuno-electron tomography (I-ET) to analyze the two- and three-dimensional structures of the late endosome–mitochondria contact sites in placental cells. Filamentous structures or tethers were identified that connected the late endosomes and mitochondria. Lamp1 antibody-labeled I-ET revealed enrichment of tethers in the MCSs. The cholesterol-binding endosomal protein metastatic lymph node 64 (MLN64) encoded by STARD3 was required for the formation of this apposition. The distance of the late endosome–mitochondria contact sites was <20 nm, shorter than that in STARD3-knockdown cells (<150 nm). The perturbation of cholesterol egress from the endosomes induced by U18666A treatment produced a longer distance in the contact sites than that in knockdown cells. The late endosome–mitochondria tethers failed to form correctly in STARD3-knockdown cells. Our results unravel the role of MLN64 involved in MCSs between late endosomes and mitochondria in placental cells.

Molecular Dynamics Simulations of the Proteins Regulating Synaptic Vesicle Fusion

Neuronal transmitters are packaged in synaptic vesicles (SVs) and released by the fusion of SVs with the presynaptic membrane (PM). An inflow of Ca2+ into the nerve terminal triggers fusion, and the SV-associated protein Synaptotagmin 1 (Syt1) serves as a Ca2+ sensor. In preparation for fusion, SVs become attached to the PM by the SNARE protein complex, a coiled-coil bundle that exerts the force overcoming SV-PM repulsion. A cytosolic protein Complexin (Cpx) attaches to the SNARE complex and differentially regulates the evoked and spontaneous release components. It is still debated how the dynamic interactions of Syt1, SNARE proteins and Cpx lead to fusion. This problem is confounded by heterogeneity in the conformational states of the prefusion protein-lipid complex and by the lack of tools to experimentally monitor the rapid conformational transitions of the complex, which occur at a sub-millisecond scale. However, these complications can be overcome employing molecular dynamics (MDs), a computational approach that enables simulating interactions and conformational transitions of proteins and lipids. This review discusses the use of molecular dynamics for the investigation of the pre-fusion protein-lipid complex. We discuss the dynamics of the SNARE complex between lipid bilayers, as well as the interactions of Syt1 with lipids and SNARE proteins, and Cpx regulating the assembly of the SNARE complex.

Comparing the Effects of Encapsulated and Non-Encapsulated Propolis Extracts on Model Lipid Membranes and Lactic Bacteria, with Emphasis on the Synergistic Effects of Its Various Compounds.

Propolis is a resinous compound made by bees with well-known biological activity. However, comparisons between encapsulated and non-encapsulated propolis are lacking. Therefore, the antibacterial activity, effect on the phase transition of lipids, and inhibition of UV-induced lipid oxidation of the two forms of propolis were compared. The results showed that non-encapsulated propolis produces quicker effects, thus being better suited when more immediate effects are required (e.g., antibacterial activity). In order to gain an in-depth introspective on these effects, we further studied the synergistic effect of propolis compounds on the integrity of lipid membranes. The knowledge of component synergism is important for the understanding of effective propolis pathways and for the perspective of modes of action of synergism between different polyphenols in various extracts. Thus, five representative molecules, all previously isolated from propolis (chrysin, quercetin, trans-ferulic acid, caffeic acid, (-)-epigallocatechin-3-gallate) were mixed, and their synergistic effects on lipid bilayers were investigated, mainly using DSC. The results showed that some compounds (quercetin, chrysin) exhibit synergism, whereas others (caffeic acid, t-ferulic acid) do not show any such effects. The results also showed that the synergistic effects of mixtures composed from several different compounds are extremely complex to study, and that their prediction requires further modeling approaches.

Roles for inter‐organelle contacts in organizing metabolism

When faced with nutrient shortage, cells adapt by remodeling their metabolic pathways and organelles. I will discuss our work using budding yeast to dissect how nutrient shortage impacts the spatial organization of metabolism and lipid droplets. We find that in response to glucose restriction, yeast remodel their mevalonate pathway by spatially compartmentalizing its rate‐limiting enzyme, HMG‐CoA Reductase (HMGCR). HMGCR spatial compartmentalization occurs at a unique inter‐organelle contact site called the nucleus‐vacuole junction (NVJ). This spatial partitioning enhances HMGCR activity, driving mevalonate synthesis to enable cellular adaptation. Our work suggests a new use for an inter‐organelle contact site in the fine‐tuning of mevalonate metabolism during nutrient stress.Remarkably, we also find that glucose restriction drives the phase transition of lipids within lipid droplets (LDs), causing them to convert from a disordered to liquid‐crystalline phase. Mechanistically, we find that these liquid crystalline lattices (LCLs) within LDs require triglyceride lipolysis. We also find that LCL‐LDs exhibit changes to the LD surface proteome. Several known LD proteins redistribute from LDs to the ER network, whereas others remain on LCL‐LDs, suggesting phase transitions of LD lipids influences LD protein targeting. Global proteomics also reveals that triglycerides harvested from LDs fuel cellular energetics at peroxisomes and mitochondria. This indicates that glucose starvation induces inter‐organelle lipid flux while promoting lipid phase transitions within LDs.

Determinants of Lipid Domain Size.

Lipid domains less than 200 nm in size may form a scaffold, enabling the concerted function of plasma membrane proteins. The size-regulating mechanism is under debate. We tested the hypotheses that large values of spontaneous monolayer curvature are incompatible with micrometer-sized domains. Here, we used the transition of photoswitchable lipids from their cylindrical conformation to a conical conformation to increase the negative curvature of a bilayer-forming lipid mixture. In contrast to the hypothesis, pre-existing micrometer-sized domains did not dissipate in our planar bilayers, as indicated by fluorescence images and domain mobility measurements. Elasticity theory supports the observation by predicting the zero free energy gain for splitting large domains into smaller ones. It also indicates an alternative size-determining mechanism: The cone-shaped photolipids reduce the line tension associated with lipid deformations at the phase boundary and thus slow down the kinetics of domain fusion. The competing influence of two approaching domains on the deformation of the intervening lipids is responsible for the kinetic fusion trap. Our experiments indicate that the resulting local energy barrier may restrict the domain size in a dynamic system.

Amyloid-beta peptide (25\u201335) triggers a reorganization of lipid membranes driven by temperature changes

The amyloid-beta peptide (Aβ) is considered a key factor in Alzheimer's disease (AD) ever since the discovery of the disease. The understanding of its damaging influence has however shifted recently from large fibrils observed in the inter-cellular environment to the small oligomers interacting with a cell membrane. We studied the effect of temperature on the latter interactions by evaluating the structural characteristics of zwitterionic phosphatidylcholine (PC) membranes with incorporated Aβ25–35 peptide. By means of small angle neutron scattering (SANS), we have observed for the first time a spontaneous reformation of extruded unilamellar vesicles (EULVs) to discoidal bicelle-like structures (BLSs) and small unilamellar vesicles (SULVs). These changes in the membrane self-organization happen during the thermodynamic phase transitions of lipids and only in the presence of the peptide. We interpret the dramatic changes in the membrane's overall shape with parallel changes in its thickness as the Aβ25–35 triggered membrane damage and a consequent reorganization of its structure. The suggested process is consistent with an action of separate peptides or small size peptide oligomers rather than the result of large Aβ fibrils.

Plant Alkaloids Inhibit Membrane Fusion Mediated by Calcium and Fragments of MERS-CoV and SARS-CoV/SARS-CoV-2 Fusion Peptides.

To rationalize the antiviral actions of plant alkaloids, the ability of 20 compounds to inhibit calcium-mediated fusion of lipid vesicles composed of phosphatidylglycerol and cholesterol was investigated using the calcein release assay and dynamic light scattering. Piperine, tabersonine, hordenine, lupinine, quinine, and 3-isobutyl-1-methylxanthine demonstrated the most potent effects (inhibition index greater than 50%). The introduction of phosphatidylcholine into the phosphatidylglycerol/cholesterol mixture led to significant changes in quinine, hordenine, and 3-isobutyl-1-methylxanthine efficiency. Comparison of the fusion inhibitory ability of the tested alkaloids, and the results of the measurements of alkaloid-induced alterations in the physical properties of model membranes indicated a potent relationship between a decrease in the cooperativity of the phase transition of lipids and the ability of alkaloids to prevent calcium-mediated vesicle fusion. In order to use this knowledge to combat the novel coronavirus pandemic, the ability of the most effective compounds to suppress membrane fusion induced by fragments of MERS-CoV and SARS-CoV/SARS-CoV-2 fusion peptides was studied using the calcein release assay and confocal fluorescence microscopy. Piperine was shown to inhibit vesicle fusion mediated by both coronavirus peptides. Moreover, piperine was shown to significantly reduce the titer of SARS-CoV2 progeny in vitro in Vero cells when used in non-toxic concentrations.

Dynamics properties of membrane proteins in native cell membranes revealed by solid-state NMR spectroscopy

Cell membranes provide an environment that is essential to the functions of membrane proteins. Cell membranes are mainly composed of proteins and highly diverse phospholipids. The influence of diverse lipid compositions of native cell membranes on the dynamics of the embedded membrane proteins has not been examined. Here we employ solid-state NMR to investigate the dynamics of E. coli Aquaporin Z (AqpZ) in its native inner cell membranes, and reveal the influence of diverse lipid compositions on the dynamics of AqpZ by comparing it in native cell membranes to that in POPC/POPG bilayers. We demonstrate that the dynamic rigidity of AqpZ generally conserves in both native cell membranes and POPC/POPG bilayers, due to its tightly packed tetrameric structure. In the gel and the liquid crystal phases of lipids, our experimental results show that AqpZ is more dynamic in native cell membranes than that in POPC/POPG bilayers. In addition, we observe that phase transitions of lipids in native membranes are less sensitive to temperature variations compared with that in POPC/POPG bilayers, which results in that the dynamics of AqpZ is less affected by the phase transitions of lipids in native cell membranes than that in POPC/POPG bilayers. This study provides new insights into the dynamics of membrane proteins in native cell membranes.

Skin hydration as a tool to control the distribution and molecular effects of intermediate polarity compounds in intact stratum corneum

The barrier function of the skin is mainly assured by its outermost layer, stratum corneum (SC), which consists of dead keratin-filled cells embedded in a lipid matrix. The skin is daily exposed to an environment with changing conditions in terms of hydration and different chemicals. Here we investigate how a molecule that has reasonable solubility in both hydrophobic and hydrophilic environments can be directed to certain regions in SC by changing the skin hydration. We use 1,2,3-trimethoxy propane (TMP) as a model substance and solid-state NMR on natural abundance 13C to obtain atomically resolved information on the molecular dynamics of TMP as well as SC lipid and protein components at varying hydration conditions. Upon dehydration, TMP redistributes from the hydrophilic corneocytes to the hydrophobic SC lipid regions. In this way, TMP can act to prevent the fluid–solid lipid transition in drying conditions and be present in the corneocytes in more humid conditions. Hydration can thereby be used as a switch to control the location and action of TMP or similar compounds in complex materials like SC. The general principles described here can also have impact on other applications including lipid-based formulations in food, drug delivery and cosmetics.

Limits of temperature adaptation and thermopreferendum

BackgroundManaging the limits of temperature adaptation is relevant both in medicine and in biotechnology. There are numerous scattered publications on the identification of the temperature limits of existence for various organisms and using different methods. Dmitry Petrovich Kharakoz gave a general explanation for many of these experimental results. The hypothesis implied that each cycle of synaptic exocytosis includes reversible phase transitions of lipids of the presynaptic membrane due to the entry and subsequent removal of calcium ions from the synaptic terminal. The correspondence of the times of phase transitions has previously been experimentally shown on isolated lipids in vitro. In order to test the hypothesis of D.P. Kharakoz in vivo, we investigated the influence of the temperature of long-term acclimatization on the temperature of heat and cold shock, as well as on the kinetics of temperature adaptation in zebrafish. Testing the hypothesis included a comparison of our experimental results with the results of other authors obtained on various models from invertebrates to humans.ResultsThe viability polygon for Danio rerio was determined by the minimum temperature of cold shock (about 6 °C), maximum temperature of heat shock (about 43 °C), and thermopreferendum temperature (about 27 °C). The ratio of the temperature range of cold shock to the temperature range of heat shock was about 1.3. These parameters obtained for Danio rerio describe with good accuracy those for the planarian Girardia tigrina, the ground squirrel Sermophilus undulatus, and for Homo sapiens.ConclusionsThe experimental values of the temperatures of cold shock and heat shock and the temperature of the thermal preferendum correspond to the temperatures of phase transitions of the lipid-protein composition of the synaptic membrane between the liquid and solid states. The viability range for zebrafish coincides with the temperature range, over which enzymes function effectively and also coincides with the viability polygons for the vast majority of organisms. The boundaries of the viability polygon are characteristic biological constants. The viability polygon of a particular organism is determined not only by the genome, but also by the physicochemical properties of lipids that make up the membrane structures of synaptic endings. The limits of temperature adaptation of any biological species are determined by the temperature range of the functioning of its nervous system.

Temperature-Sensitive Lipid-Coated Carbon Nanotubes for Synergistic Photothermal Therapy and Gene Therapy.

The combination of photothermal therapy (PTT) and gene therapy (GT) shows great potential to achieve synergistic anti-tumor activity. However, the lack of a controlled release of genes from carriers remains a severe hindrance. Herein, peptide lipid (PL) and sucrose laurate (SL) were used to coat single-walled carbon nanotubes (SCNTs) and multi-walled carbon nanotubes (MCNTs) to form bifunctional delivery systems (denoted SCNT-PS and MCNT-PS, respectively) with excellent temperature-sensitivity and photothermal performance. CNT/siRNA suppressed tumor growth by silencing survivin expression while exhibiting photothermal effects under near-infrared (NIR) light. SCNT-PS/siRNA showed very high anti-tumor activity, resulting in the complete inhibition of some tumors. It was highly efficient for systemic delivery to tumor sites and to facilitate siRNA release owing to the phase transition of the temperature-sensitive lipids, due to PL and SL coating. Thus, SCNT-PS/siRNA is a promising anti-tumor nanocarrier for combined PTT and GT.

Recent Progress to Explore the Mechanism of Direct Internalization of Cell-Penetrating Peptide (CPP) through BioMembrane

A specific series of peptides, called a cell-penetrating peptide (CPP), is known to be free to directly permeate through cell membranes into the cytosol (cytolysis); hence, this CPP would be a potent carrier for a drug delivery system (DDS). Previously, we proposed the mechanism of cytolysis as a temporal and local phase transfer of membrane lipid caused by positive membrane curvature generation. Moreover, we showed how to control the CPP cytolysis based on the mechanism. Here, we introduce key factors effectively modulate the CPC cytolysis so that they could be potent tools to control cytolysis. This short communication gives overview of our progressive investigation on the CPC cytolysis mechanism which inspired us to imagine many ways to control the cytolysis either to enhance or to suppress by maneuvering membrane curvatures corresponding to the temporal phase transitions of self-assembled membrane lipids under the liquid crystal states. We have shown the usefulness of physical stimuli such as osmotic pressure or particle size and chemical stimuli with their Hoffmeister effect to the outer medium by changing the hydration level of outer layers of membrane to control the CPP cytolysis by changing membrane curvature. Taking all the revealed vital factors and their effects as a tool, we will further explore how to control CPP cytolysis for developing a DDS system combined with appropriate cargo selection to be tagged with CPPs.

Effects of the water-soluble fraction of the mixture fuel oil/diesel on the microalgae Dunaliella tertiolecta through growth.

There is evidence that water-soluble fraction (WSF) from fuel oil/diesel mixture affects marine microbiota. In order to establish a sequence of WSF effects during microalgal growth, this work aimed to monitor Dunaliella tertiolecta exposed to WSF during 15days. Three different pigments (chlorophyll a, lutein, and β-carotene) and four metabolites (protein, lipids, fatty acids, and phenols) were studied, and FTIR spectroscopy was used to determine the biomolecular transitions of lipids and their accumulation. The results show that D. tertiolecta triggered a physiological and biochemical response with changes in growth rate, pigments, phenols, lipids, and proteins of the microalga, although fatty acid profile was unaltered. For all the biochemical parameters altered, there were significant differences with the controls. At the end of the assay, exposed D. tertiolecta showed similar values with the control on all the compounds analyzed, except lipids. FTIR absorbance showed an increase in unsaturated acyl chains within the exposed microalgae, giving support for a possible uptake of hydrocarbons from WSF. Variation in pigments and phenol contents is presented as an integrated antioxidant response to the stress imposed by WSF. Overall, this research provides information about the effects of WSF on D. tertiolecta, and the ability of this microalga to recover after long-term exposure to the water-soluble fraction of fuel oil/diesel.

Mechanical Properties Determination of DMPC, DPPC, DSPC, and HSPC Solid-Ordered Bilayers

Lipid bilayers are active participants in many crucial biological processes. They can be observed in different phases, liquid and solid, respectively. The liquid phase is predominant in biological systems. The solid phase, both crystalline and gel phases, is under investigation due to its resilience to mechanical stress and tight packing of lipids. The mechanical properties of lipids affect their dynamics, therefore influencing the transformation of cell plasma and the endomembrane. Mechanical properties of lipid bilayers are also an important parameter in the design and production of supramolecular lipid-based drug delivery systems. To this end, in this work, we focused on investigating the effect of solid phases of lipid bilayers on their structural parameters and mechanical properties using theoretical molecular dynamics studies on atomistic models of whole vesicles. Those include area per lipid, membrane thickness, density vesicle profiles, bending rigidity coefficient, and area compressibility. Additionally, the bending rigidity coefficient was measured using the flicker noise spectroscopy. The two approaches produced very similar and consistent results. We showed that, contrary to our expectations, bending rigidity coefficients of solid-ordered bilayers for vesicles decreased with an increase in lipid transition temperature. This tendency was reverse in planar systems. Additionally, we have observed an increase of membrane thickness and area compressibility and a decrease of area per lipid. We hope these results will provide valuable mechanical insight for the behavior in solid phases and differences between spherical and planar confirmations.

Interleaflet Interaction in Phase Separated Asymmetric Lipid Bilayers

Ordered lipid domains (OLD) may serve as signaling platforms provided both membrane leaflets contribute. Interleaflet registration of phase separated regions in symmetric bilayers is driven by (i) the line tension around the thicker ordered domains (1) and (ii) membrane undulations (2, 3). How these forces act to generate membrane spanning domains in compositionally different leaflets is unknown. Here we use both fluorescence microscopy and fluorescence correlation spectroscopy of folded asymmetric planar lipid bilayers (4) to observe the enslavement of disordered lipids into ordered domains by ordered lipids in the opposing leaflet and, vice versa, the partial dissolution of ordered phases by disordered lipids in the opposing leaflet. These results were mirrored by the transition of lipids from the gel phase to the liquid phase and vice versa as seen by molecular dynamics simulations of compositionally symmetric, but - due to temperature differences - phase asymmetric bilayers. Our analysis of elastic lipid deformations, thermal membrane fluctuations and the structure of the fluid/gel boundary shows that the alignment of the original domain in one leaflet with the induced domain in the opposing leaflet is the energetically favored state and that the phase boundaries in the individual leaflets are shifted relative to each other by a few nanometers along the membrane surface. We conclude that similar forces govern domain registration in both symmetric and asymmetric membranes. 1. Galimzyanov, et al. Phys. Rev. Lett. 2015, 115:088101. 2. Horner, Antonenko, Y, Pohl. Biophys. J. 2009, 96:2689. 3. Galimzyanov, et al. Biophys. J. 2017, 112:339. 4. Horner; Akimov; Pohl. Phys. Rev. Lett. 2013, 110:268101.

Effect of the desiccation tolerance and cryopreservation methods on the viability of Citrus limon L. Burm cv. Eureka seeds

Cryopreservation of the germplasm for long-term periods is of great importance to maintain the genetic resource. Argentina is one of the world's highest lemon producing country. The performance of different cooling/warming rates in the cryopreservation method of Citrus limon L. Burm cv. Eureka seeds and their influence on the interval of optimal moisture content in the desiccation stage were analyzed. Water sorption isotherm was determined and modeled using D'Arcy & Watt equation; it provided important information concerning the amounts of water associated to strong, weak and multimolecular binding sites along the sorption isotherm. Seeds tolerated a wide range of desiccation conditions (0.1<aw<0.85) showing a high viability (>80%), however desiccation to 0.0526 g H2O g−1 d.b. (aw = 0.0901) produced a significant loss of viability. Differential Scanning Calorimetry was used to identify the thermal transitions of lipids and water in the seed; enthalpies were used to calculate the unfrozen water fraction (0.19 g H2O g−1 d.b. corresponding to aw = 0.64). Two cooling/warming rates were tested on desiccated seeds (0.11<aw<0.85): i) 200 °C min−1 (reached with seeds placed inside a closed cryogenic vial); ii) 1000 °C min−1 (reached with aluminum-foiled seeds placed in a perforated cryogenic vial). For both methods, viability was maximum (83.3%) at aw = 0.64. Lethal ice formation was responsible for the loss of viability at aw>0.64 corresponding to the unfrozen water fraction. The use of higher cooling/warming rates enables a wider range of desiccation conditions (0.33<aw<0.76) in cryopreservation procedures. This work contributes to the optimization of cryopreservation methods of economically important germplasm.