Effects of grafted polymers on the lipid membrane fluidity.

Since the membrane fluidity controls the cellular functions, it is important to identify the factors that determine the cell membrane viscosity. Cell membranes are composed of not only lipids and proteins but also polysaccharide chain-anchored molecules, such as glycolipids. To reveal the effects of grafted polymers on the membrane fluidity, in this study, we measured the membrane viscosity of polymer-grafted giant unilamellar vesicles (GUVs), which were prepared by introducing the poly (ethylene glycol) (PEG)-anchored lipids to the ternary GUVs composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol. The membrane viscosity was obtained from the velocity field on the GUV generated by applying a point force, based on the hydrodynamic model proposed by Henle and Levine. The velocity field was visualized by a motion of the circular liquid ordered (Lo) domains formed by a phase separation. With increasing PEG density, the membrane viscosity of PEG-grafted GUVs increased gradually in the mushroom region and significantly in the brush region. We propose a hydrodynamic model that includes the excluded volume effect of PEG chains to explain the increase in membrane viscosity in the mushroom region. This work provides a basic understanding of how grafted polymers affect the membrane fluidity.

Morphology of vesicle triplets: shape transformation at weak and strong adhesion limits.

We investigate the morphologies of adhering vesicle triplets as a function of volume-to-area ratio encoded by the reduced volume in strong and weak adhesion regimes. In the strong adhesion regime, the morphology change of the vesicle triplet depends on the arrangement of vesicles. By decreasing the reduced volume, a triangular triplet composed of three spherical caps with a trifurcated flat contact zone deformed to a compact spherical shape with a sigmoidal contact zone, whereas a linear vesicle triplet composed of pancake-shaped vesicles sandwiched between two spherical-cap vesicles with a flat contact zone deformed into a compact spherical shape with biconvex interfaces. The morphologies of vesicle triplets with flat contact zones are reproduced by the so-called two-tension model based on the total energy consisting of bending energy, adhesion energy and surface energy, where the surface tension in the noncontact zone is different from that in the contact zone. When the flat interface deforms, the two-tension model is no longer applicable. The compact spherical triplets with curved interfaces can be reproduced by introducing geometrical constraints requiring that the total area of the non-contact zones is minimal, thereby confining the aggregate to a spherical cavity; this is referred to as the cavity model. In the weak adhesion regime, vesicle triplets with either a triangular or linear topology deform into prolate-based triplets by decreasing the reduced volume.

Quantification of Giant Unilamellar Vesicle Fusion Products by High-Throughput Image Analysis.

Artificial cells are based on dynamic compartmentalized systems. Thus, remodeling of membrane-bound systems, such as giant unilamellar vesicles, is finding applications beyond biological studies, to engineer cell-mimicking structures. Giant unilamellar vesicle fusion is rapidly becoming an essential experimental step as artificial cells gain prominence in synthetic biology. Several techniques have been developed to accomplish this step, with varying efficiency and selectivity. To date, characterization of vesicle fusion has relied on small samples of giant vesicles, examined either manually or by fluorometric assays on suspensions of small and large unilamellar vesicles. Automation of the detection and characterization of fusion products is now necessary for the screening and optimization of these fusion protocols. To this end, we implemented a fusion assay based on fluorophore colocalization on the membranes and in the lumen of vesicles. Fluorescence colocalization was evaluated within single compartments by image segmentation with minimal user input, allowing the application of the technique to high-throughput screenings. After detection, statistical information on vesicle fluorescence and morphological properties can be summarized and visualized, assessing lipid and content transfer for each object by the correlation coefficient of different fluorescence channels. Using this tool, we report and characterize the unexpected fusogenic activity of sodium chloride on phosphatidylcholine giant vesicles. Lipid transfer in most of the vesicles could be detected after 20 h of incubation, while content exchange only occurred with additional stimuli in around 8% of vesicles.

From vesicles toward protocells and minimal cells.

In contrast to ordinary condensed matter systems, "living systems" are unique. They are based on molecular compartments that reproduce themselves through (i) an uptake of ingredients and energy from the environment, and (ii) spatially and timely coordinated internal chemical transformations. These occur on the basis of instructions encoded in information molecules (DNAs). Life originated on Earth about 4 billion years ago as self-organised systems of inorganic compounds and organic molecules including macromolecules (e.g. nucleic acids and proteins) and low molar mass amphiphiles (lipids). Before the first living systems emerged from non-living forms of matter, functional molecules and dynamic molecular assemblies must have been formed as prebiotic soft matter systems. These hypothetical cell-like compartment systems often are called "protocells". Other systems that are considered as bridging units between non-living and living systems are called "minimal cells". They are synthetic, autonomous and sustainable reproducing compartment systems, but their constituents are not limited to prebiotic substances. In this review, we focus on both membrane-bounded (vesicular) protocells and minimal cells, and provide a membrane physics background which helps to understand how morphological transformations of vesicle systems might have happened and how vesicle reproduction might be coupled with metabolic reactions and information molecules. This research, which bridges matter and life, is a great challenge in which soft matter physics, systems chemistry, and synthetic biology must take joined efforts to better understand how the transformation of protocells into living systems might have occurred at the origin of life.

An autopsied case report of spastic paraplegia with thin corpus callosum carrying a novel mutation in the SPG11 gene: widespread degeneration with eosinophilic inclusions.

BACKGROUND: The detailed neuropathological features of patients with autosomal recessive hereditary spastic paraplegia with a thin corpus callosum (TCC) and SPG11 mutations are poorly understood, as only a few autopsies have been reported. Herein, we describe the clinicopathological findings of a patient with this disease who received long-term care at our medical facility. CASE PRESENTATION: A Japanese man exhibited a mild developmental delay in early childhood and intellectual disability, followed by the appearance of a spastic gait by age 13. At the age of 25 years, he became bedridden and needed a ventilator. Genetic analysis revealed a homozygous splice site variant in the SPG11 gene (c. 4162-2A > G) after the provision of genetic counselling and acquisition of informed consent from his parents. He died of pneumonia at the age of 44. His brain weighed 967 g and was characterized by a TCC, and his spinal cord was flattened. Microscopically, degeneration was observed in the posterior spinocerebellar tract, the gracile fasciculus, and the posterior column in addition to the corticospinal tract. Marked neuronal loss and gliosis were observed in the anterior horn, Clarke's column, and hypoglossal and facial nuclei. Various types of neurons, in addition to motor neurons, showed coarse eosinophilic granules that were immunoreactive for p62. The loss of pigmented neurons with gliosis was apparent in both the substantia nigra and locus coeruleus. Lateral geniculate body degeneration was a characteristic feature of this patient. Furthermore, peripheral Lewy body-related α-synucleinopathy and scattered α-synuclein-immunoreactive neurites in the locus coeruleus and reticular formation of the brainstem were observed. CONCLUSIONS: In patients with hereditary spastic paraplegia with SPG11 mutations, a variety of clinical phenotypes develop due to widespread lesions containing p62-immunoreactive neuronal cytoplasmic inclusions. We herein report the lateral geniculate body as another degenerative site related to SPG11-related pathologies that should be studied in future investigations.

Vesicle deformation and division induced by flip-flops of lipid molecules.

We investigated the deformation of small unilamellar vesicles (SUVs) induced by flip-flops of lipids using coarse-grained molecular dynamics simulations. In the case of single-component SUVs composed of zero spontaneous curvature lipids (ZLs), the flip-flop of ZLs deformed stomatocyte-shaped SUVs into an oblate shape, whereas pear-shaped SUVs were deformed into a prolate shape. These two equilibrium shapes comply with the local minima of elastic energy. In the case of binary vesicles composed of ZLs and negative spontaneous curvature lipids (NLs), the vesicle deformation pathway depended on the initial NL distribution in the bilayer. If the initial difference in the NL concentration between the outer and inner leaflets was small, the flip-flop of ZLs and NLs rapidly deformed pear-shaped SUVs into an equilibrium prolate shape. On the other hand, when NLs were localised in the inner leaflet, the flip-flop of ZLs and NLs deformed pear-shaped SUVs into a limiting shape and then induced vesicle division. Because the flip-flop rate of NLs is much faster than that of ZLs, the total free energy was first relaxed by the flip-flop of NLs and then by that of ZLs. This kinetic effect is responsible for the observed vesicle division induced by flip-flops.

Viscosity Landscape of Phase-Separated Lipid Membrane Estimated from Fluid Velocity Field.

In cell membranes, the functional constituents such as peptides, proteins, and polysaccharides diffuse in a sea of lipids as single molecules and molecular aggregates. Thus, the fluidity of the heterogeneous multicomponent membrane is important for understanding the roles of the membrane in cell functionality. Recently, Henle and Levine described the hydrodynamics of molecular diffusion in a spherical membrane. A tangential point force at the north pole induces a pair of vortices whose centers lie on a line perpendicular to the point force and are symmetrical with respect to the point force. The position of the vortex center depends on ηm/Rηw, where R is the radius of the spherical membrane, and ηm and ηw are the viscosities of the membrane and the surrounding medium, respectively. Based on this theoretical prediction, we applied a point force to a phase-separated spherical vesicle composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol by means of a microinjection technique. The pathlines were visualized by trajectories of microdomains. We determined the position of the vortex center and estimated the membrane viscosity using the dependence of the position of the vortex center on ηm/Rηw. The obtained apparent membrane viscosities for various compositions are mapped on the phase diagram. The membrane viscosity is almost constant in the range of 0 <ϕLo ≤ 0.5 (ϕLo: area fraction of the liquid ordered phase), whereas that in the range of 0.5 ≤ ϕLo < 1.0 exponentially increases with increase of ϕLo. The obtained viscosity landscape provides a basic understanding of the fluidity of heterogeneous multicomponent membranes.

Morphologies of Vesicle Doublets: Competition among Bending Elasticity, Surface Tension, and Adhesion.

To study the mechanical laws governing the form of multicellular organisms, we examine the morphology of adhering vesicle doublets as the simplest model system. We monitor the morphological transformations of doublets induced by changes of adhesion strength and volume/area ratio, which are controlled by intermembrane interactions and thermal area expansion, respectively. When we increase the temperature in the weak adhesion regime, a dumbbell flat-contact doublet is transformed to a parallel-prolate doublet, whereas in the strong adhesion regime, heating transforms the dumbbell flat-contact doublet into a spherical sigmoid-contact doublet. We reproduce the observed doublet morphologies by numerically minimizing the total energy, including the contact-potential adhesion term as well as the surface and bending terms, using the Surface Evolver package. From the reproduced morphologies, we extract the adhesion strength, the surface tension, and the volume/area ratio of the vesicles, which reveals the detailed mechanisms of the morphological transitions in doublets.

Migration of Deformable Vesicles Induced by Ionic Stimuli.

We have investigated the dynamics of phospholipid vesicles composed of 1,2-dioleoyl- sn-glycero-3-phosphocholine triggered by ionic stimuli using electrolytes such as CaCl2, NaCl, and NaOH. The ionic stimuli induce two characteristic vesicle dynamics, deformation due to the ion binding to the lipids in the outer leaflet of the vesicle and migration due to the concentration gradient of ions, that is, diffusiophoresis or the interfacial energy gradient mechanism. We examined the deformation pathway for each electrolyte as a function of time and analyzed it based on the surface dissociation model and the area difference elasticity model, which reveals the change of the cross-sectional area of the phospholipid by the ion binding. The metal ions such as Ca2+ and Na+ encourage inward budding deformation by decreasing the cross-sectional area of a lipid, whereas the hydroxide ion (OH-) encourages outward budding deformation by increasing the cross-sectional area of a lipid. When we microinjected these electrolytes toward the vesicles, a strong coupling between the deformation and the migration of the vesicle was observed for CaCl2 and NaOH, whereas for NaCl, the coupling was very weak. This difference probably originates from the binding constants of the ions.

Molecular mechanism of vesicle division induced by coupling between lipid geometry and membrane curvatures.

We investigated the effects of lipid geometry on vesicle division using coarse grained molecular dynamics simulations. When the vesicle is composed of zero and negative spontaneous curvature lipids (ZSLs and NSLs), the difference in their molecular spontaneous curvatures destabilizes the neck of the limiting shape vesicle. In the vesicle division pathway, the neck developed into the stalk intermediates. The stalk was broken when the NSLs were expelled from the stalk. Free energy analysis shows that the coupling between the lipid geometry and the Gaussian rigidity is responsible for the observed vesicle division.

Migration of Phospholipid Vesicles Can Be Selectively Driven by Concentration Gradients of Metal Chloride Solutions.

We have investigated the migrations of phospholipid vesicles under the concentration gradients of metal ions. We microinjected metal chloride solutions, monovalent (NaCl and KCl), divalent (CaCl2 and MgCl2), and trivalent (LaCl3) salts, toward phospholipid giant vesicles (GVs) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). For NaCl, CaCl2, and MgCl2 solutions, the GVs migrated straight toward the tip of the micropipette in response to the concentration gradients, whereas for KCl and LaCl3, GVs moved to the opposite direction. Our motion tracking of lipid domains in a vesicle membrane showed no unidirectional flow in the membrane during the vesicle migration, indicating that the Marangoni mechanism is not responsible for the observed vesicle migration. We calculated the diffusiophoretic velocities for symmetric and asymmetrical electrolytes by solving the Stokes' equation numerically. The theoretical diffusiophoretic velocities described the observed migration velocities well. Thus, we can control the migration of vesicle in response to the concentration gradient by adapting the electrolytes and the lipids.

Pharmacological evaluation of novel (6-aminopyridin-3-yl)(4-(pyridin-2-yl)piperazin-1-yl) methanone derivatives as TRPV4 antagonists for the treatment of pain.

A novel series of (6-aminopyridin-3-yl)(4-(pyridin-2-yl)piperazin-1-yl) methanone derivatives were identified as selective transient receptor potential vanilloid 4 (TRPV4) channel antagonist and showed analgesic effect in Freund's Complete Adjuvant (FCA) induced mechanical hyperalgesia model in guinea pig and rat. Modification of right part based on the compound 16d which was disclosed in our previous communication led to the identification of compound 26i as a flagship compound. In this paper, we described the details about design, synthesis and structure-activity relationship (SAR) analysis at right and left part of these derivatives (Fig. 1).

Role of Inverse-Cone-Shape Lipids in Temperature-Controlled Self-Reproduction of Binary Vesicles.

We investigate a temperature-driven recursive division of binary giant unilamellar vesicles (GUVs). During the heating step of the heating-cooling cycle, the spherical mother vesicle deforms to a budded limiting shape using up the excess area produced by the chain melting of the lipids and then splits off into two daughter vesicles. Upon cooling, the daughter vesicle opens a pore and recovers the spherical shape of the mother vesicle. Our GUVs are composed of DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine) and DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine). During each cycle, vesicle deformation is monitored by a fast confocal microscope and the images are analyzed to obtain the time evolution of reduced volume and reduced monolayer area difference as the key geometric parameters that quantify vesicle shape. By interpreting the deformation pathway using the area-difference elasticity theory, we conclude that vesicle division relies on (1) a tiny asymmetric distribution of DLPE within the bilayer, which controls the observed deformation from the sphere to the budded shape; and (2) redistribution of DLPE during the deformation-division stage, which ensures that the process is recursive. The spontaneous coupling between membrane curvature and PE lipid distribution is responsible for the observed recursive division of GUVs. These results shed light on the mechanisms of vesicle self-reproduction.

Migration of phospholipid vesicles in response to OH(-) stimuli.

We demonstrate migration of phospholipid vesicles in response to a pH gradient. Upon simple micro-injection of a NaOH solution, the vesicles linearly moved to the tip of the micro-pipette and the migration velocity was proportional to the gradient of OH(-) concentration. Vesicle migration was characteristic of OH(-) ions and no migration was observed for monovalent salts or nonionic sucrose solutions. The migration of vesicles is quantitatively described by the surface tension gradient model where the hydrolysis of the phospholipids by NaOH solution decreases the surface tension of the vesicle. The vesicles move toward a direction where the surface energy decreases. Thus the chemical modification of lipids produces a mechanical force to drive vesicles.

Discovery of novel 2',4'-dimethyl-[4,5'-bithiazol]-2-yl amino derivatives as orally bioavailable TRPV4 antagonists for the treatment of pain: Part 2.

A series of 2',4'-dimethyl-[4,5'-bithiazol]-2-yl amino derivatives have been identified as selective TRPV4 antagonists that display inhibition potencies against 4α-phorbol 12,13-didecanoate (4αPDD), well known as a TRPV4 selective agonist and/or a hypotonicity. In particular, 9-(6-((2',4'-dimethyl-[4,5'-bithiazol]-2-yl)amino)nicotinoyl)-3-oxa-9-azabicyclo[3.3.1]nonan-7-one showed an analgesic effect in Freund's Complete Adjuvant (FCA) induced mechanical hyperalgesia model in guinea pig (reported in Part 1). However, there are some concerns such as species differences and the need for higher plasma exposure to achieve target efficacy for evaluation by an in vivo pain model. In this Letter, we report the resolution of some of the problems by further optimizing the chemical scaffold.

Discovery of novel 2',4'-dimethyl-[4,5'-bithiazol]-2-yl amino derivatives as orally bioavailable TRPV4 antagonists for the treatment of pain: Part 1.

A novel series of 2',4'-dimethyl-[4,5'-bithiazol]-2-yl amino derivatives were found by high throughput screening of the TRPV4 receptor, at which these compounds showed competitive antagonist potential against 4α-phorbol 12,13-didecanoate (4αPDD) as the selective TRPV4 agonist and showed excellent selectivity for TRPV1, N-type and L-type calcium ion channels, but poor ADME profile. In our SAR strategy, we found that the lead molecule 1 also having the unique 3-oxa-9-azabicyclo [3.3.1] nonan-7-one on the right part showed potent TRPV4 antagonist activity, good solubility at pH 6.8, good microsomal stability for human and better ADME profile including oral bioavailability. Moreover, compound 1 had an analgesic effect in Freund's Complete Adjuvant (FCA) induced mechanical hyperalgesia model in guinea pig. In this letter, we report a lead optimization process to identify the lead compound 1 (Fig. 1).

[Safety and efficacy of surgical closure of the larynx for recurrent aspiration pneumonia in persons with severe motor and intellectual disabilities: a comparative study with tracheoesophageal diversion].

OBJECTIVE: We retrospectively investigated the efficacy and complications of surgical closure of the larynx (SCL) for recurrent aspiration pneumonia in comparison with tracheoesophageal diversion. METHODS: The subjects were persons with severe motor and intellectual disabilities (SMID) who had undergone surgery for recurrent aspiration pneumonia between 1994 and 2011: A 8 SCL patients group and a 16 tracheoesophageal diversion patients group. We investigated two groups the lower respiratory infection incidence, length of hospital stay for the surgery, postoperative complications, and rate of cannula withdrawal, by reviewing medical records. RESULTS: Both the SCL and the tracheoesophageal diversion group showed a reduction in the incidence of infection after surgery, indicating that the efficacy of SCL was equivalent to that of tracheoesophageal diversion in preventing aspiration pneumonea. The SCL group showed a reduction in the length of hospital stay and an increased rate of cannula withdrawal as compared with the tracheoesophageal diversion group. CONCLUSION: The efficacy of SCL was equivalent to that of tracheoesophageal diversion in preventing aspiration for SMID. We consider SLC to have potential for reducing the burden on patients.

Shape transformations of toroidal vesicles.

Morphologies of genus-1 and 2 toroidal vesicles are studied numerically by dynamically triangulated membrane models and experimentally by confocal laser microscopy. Our simulation results reproduce shape transformations observed in our experiments well. At large reduced volumes of the genus-1 vesicles, obtained vesicle shapes agree with the previous theoretical prediction, in which axisymmetric shapes are assumed: double-necked stomatocyte, discoidal toroid, and circular toroid. However, for small reduced volumes, it is revealed that a non-axisymmetric discoidal toroid and handled discocyte exist in thermal equilibrium in the parameter range, in which the previous theory predicts axisymmetric discoidal shapes. Polygonal toroidal vesicles and subsequent budding transitions are also found. The entropy caused by shape fluctuations slightly modifies the stability of the vesicle shapes.

Dynamics of fatty acid vesicles in response to pH stimuli.

We investigate the dynamics of decanoic acid/decanoate (DA) vesicles in response to pH stimuli. Two types of dynamic processes induced by the micro-injection of NaOH solutions are sequentially observed: deformations and topological transitions. In the deformation stage, DA vesicles show a series of shape deformations, i.e., prolate-oblate-stomatocyte-sphere. In the topological transition stage, spherical DA vesicles follow either of the two pathways, pore formation and vesicle fusion. The pH stimuli modify a critical aggregation concentration of DA molecules, which causes the solubilization of DA molecules in the outer leaflet of the vesicle bilayers. This solubilization decreases the outer surface area of the vesicle, thereby increasing surface tension. A kinetic model based on area difference elasticity theory can accurately describe the dynamics of DA vesicles triggered by pH stimuli.

Concepts of a synthetic minimal cell: Information molecules, metabolic pathways, and vesicle reproduction.

How do the living systems emerge from non-living molecular assemblies? What physical and chemical principles supported the process? To address these questions, a promising strategy is to artificially reconstruct living cells in a bottom-up way. Recently, the authors developed the "synthetic minimal cell" system showing recursive growth and division cycles, where the concepts of information molecules, metabolic pathways, and cell reproduction were artificially and concisely redesigned with the vesicle-based system. We intentionally avoided using the sophisticated molecular machinery of the biological cells and tried to redesign the cells in the simplest forms. This review focuses on the similarities and differences between the biological cells and our synthetic minimal cell concerning each concept of cells. Such comparisons between natural and artificial cells will provide insights on how the molecules should be assembled to create living systems to the wide readers in the field of synthetic biology, artificial cells, and protocells research. This review article is an extended version of the Japanese article "Growth and division of vesicles coupled with information molecules," published in SEIBUTSU-BUTSURI vol. 61, p. 378-381 (2021).