Osmotic Pressure Enables High-Yield Assembly of Giant Vesicles in Solutions of Physiological Ionic Strengths.

Giant unilamellar vesicles (GUVs) are micrometer-scale minimal cellular mimics that are useful for bottom-up synthetic biology and drug delivery. Unlike assembly in low-salt solutions, assembly of GUVs in solutions with ionic concentrations of 100-150 mM Na/KCl (salty solutions) is challenging. Chemical compounds deposited on the substrate or incorporated into the lipid mixture could assist in the assembly of GUVs. Here, we investigate quantitatively the effects of temperature and chemical identity of six polymeric compounds and one small molecule compound on the molar yields of GUVs composed of three different lipid mixtures using high-resolution confocal microscopy and large data set image analysis. All the polymers moderately increased the yields of GUVs either at 22 or 37 °C, whereas the small molecule compound was ineffective. Low-gelling temperature agarose is the singular compound that consistently produces yields of GUVs of greater than 10%. We propose a free energy model of budding to explain the effects of polymers in assisting the assembly of GUVs. The osmotic pressure exerted on the membranes by the dissolved polymer balances the increased adhesion between the membranes, thus reducing the free energy for bud formation. Data obtained by modulating the ionic strength and ion valency of the solution shows that the evolution of the yield of GUVs supports our model's prediction. In addition, polymer-specific interactions with the substrate and the lipid mixture affects yields. The uncovered mechanistic insights provide a quantitative experimental and theoretical framework to guide future studies. Additionally, this work shows a facile means for obtaining GUVs in solutions of physiological ionic strengths.

Identification and functional characterization of two novel mutations in KCNJ10 and PI4KB in SeSAME syndrome without electrolyte imbalance.

BACKGROUND: Dysfunction in inwardly rectifying potassium channel Kir4.1 has been implicated in SeSAME syndrome, an autosomal-recessive (AR), rare, multi-systemic disorder. However, not all neurological, intellectual disability, and comorbid phenotypes in SeSAME syndrome can be mechanistically linked solely to Kir4.1 dysfunction. METHODS: We therefore performed whole-exome sequencing and identified additional genetic risk-elements that might exert causative effects either alone or in concert with Kir4.1 in a family diagnosed with SeSAME syndrome. RESULTS: Two variant prioritization pipelines based on AR inheritance and runs of homozygosity (ROH), identified two novel homozygous variants in KCNJ10 and PI4KB and five rare homozygous variants in PVRL4, RORC, FLG2, FCRL1, NIT1 and one common homozygous variant in HSPA6 segregating in all four patients. The novel mutation in KCNJ10 resides in the cytoplasmic domain of Kir4.1, a seat of phosphatidylinositol bisphosphate (PIP2) binding. The mutation altered the subcellular localization and stability of Kir4.1 in patient-specific lymphoblastoid cells (LCLs) compared to parental controls. Barium-sensitive endogenous K+ currents in patient-specific LCLs using whole-cell patch-clamp electrophysiology revealed membrane depolarization and defects in inward K+ ion conductance across the membrane, thereby suggesting a loss-of-function effect of KCNJ10 variant. CONCLUSION: Altogether, our findings implicate the role of new genes in SeSAME syndrome without electrolyte imbalance and thereby speculate the regulation of Kir4.1 channel activity by PIP2 and integrin-mediated adhesion signaling mechanisms.

Size Distributions and Yields of Giant Vesicles Assembled on Cellulose Papers and Cotton Fabric.

Lamellar phospholipid stacks on cellulose paper vesiculate to form cell-like giant unilamellar vesicles (GUVs) in aqueous solutions. The sizes and yields of the GUVs that result and their relationship to the properties of the cellulose fibers are unknown. Here, we report the characteristics of GUVs produced on four different cellulose substrates, three disordered porous media consisting of randomly entangled cellulose fibers (high-purity cellulose filter papers of different effective porosities), and an ordered network of weaved cellulose fibers (cotton fabric). Large numbers of GUVs formed on all four substrates. This result demonstrates for the first time that GUVs form on cotton fabric. Despite differences in the effective porosities and the configuration of the cellulose fibers, all four substrates yielded populations of GUVs with similar distribution of diameters. The distribution of diameters of the GUVs had a single well-defined peak and a right tail. Ninety-eight percent of the GUVs had diameters less than the average diameter of the cellulose fibers (∼20 micrometers). Cotton fabric produced the highest yield of GUVs with the lowest sample-to-sample variation. Moreover, cotton fabric is reusable. Fabric used sequentially produced similar crops of GUVs at each cycle. At the end of the sequence, there was no apparent change in the cellulose fibers. Cellulose fibers thus promote the vesiculation of lamellar phospholipid stacks in aqueous solutions.

Fabrics of Diverse Chemistries Promote the Formation of Giant Vesicles from Phospholipids and Amphiphilic Block Copolymers.

Giant vesicles composed of phospholipids and amphiphilic block copolymers are useful for biomimetic drug delivery, for biophysical experiments, and for creating synthetic cells. Here, we report that large numbers of giant unilamellar vesicles (GUVs) can be formed on a broad range of fabrics composed of entangled cylindrical fibers. We show that fabrics woven from fibers of silk, wool, rayon, nylon, polyester, and fiberglass promote the formation of GUVs and giant polymer vesicles (polymersomes) in aqueous solutions. The result extends significantly previous reports on the formation of GUVs on cellulose paper and cotton fabric. Giant vesicles formed on all the fabrics from lipids with various headgroup charges, chains lengths, and chain saturations. Giant vesicles could be formed from multicomponent lipid mixtures, from extracts of plasma membranes, and from amphiphilic diblock and triblock copolymers, in both low ionic strength and high ionic strength solutions. Intriguingly, statistical characterization using a model lipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine, revealed that the majority of the fabrics yielded similar average counts of vesicles. Additionally, the vesicle populations obtained from the different fabrics had similar distributions of sizes. Fabrics are ubiquitous in society in consumer, technical, and biomedical applications. The discovery herein that biomimetic GUVs grow on fabrics opens promising new avenues in vesicle-based smart materials design.

Cellulose Abetted Assembly and Temporally Decoupled Loading of Cargo into Vesicles Synthesized from Functionally Diverse Lamellar Phase Forming Amphiphiles.

Self-assembled micrometer-scale vesicles composed of lamellar phase forming amphiphiles are useful as chemical microreactors, as minimal artificial cells, as protocell mimics for studies of the origins of life, and as vehicles for the targeted delivery of drugs. Given their varied uses, discovery of a universal mechanism that is simple, rapid, and that produces vesicles from a large variety of amphiphiles with different chemical and physical properties at high yield is extremely desirable. Here we show that cellulose, in the form of cellulose paper, facilitates the assembly of membranous vesicles 5-20 µm in diameter from scientifically and technologically important amphiphiles of diverse chemical structures and functionality such as fatty acids (fatty acid vesicles), amphiphilic diblock copolymers, and amphiphilic triblock copolymers (polymersomes). Assembly of vesicles occurred within 90 min of placing the amphiphile-coated cellulose paper into aqueous solutions. Varying thermal and chemical conditions, however, are required for the high-yield assembly of vesicles from the different amphiphiles. The vesicles, when attached to cellulose fibers, have membranes that remain unsealed. This topological characteristic of the vesicles grown on paper allowed the scalable separation of the process of growth from the process of loading cargo (temporally decoupled growth and loading). We demonstrate a temporally decoupled process to rapidly produce large quantities of protein-loaded polymersomes on the benchtop by using high temperatures to accelerate the growth of the polymersomes and subsequently milder temperatures during diffusive loading of the protein cargo.

Noncontact orientation of objects in three-dimensional space using magnetic levitation.

This paper describes several noncontact methods of orienting objects in 3D space using Magnetic Levitation (MagLev). The methods use two permanent magnets arranged coaxially with like poles facing and a container containing a paramagnetic liquid in which the objects are suspended. Absent external forcing, objects levitating in the device adopt predictable static orientations; the orientation depends on the shape and distribution of mass within the objects. The orientation of objects of uniform density in the MagLev device shows a sharp geometry-dependent transition: an analytical theory rationalizes this transition and predicts the orientation of objects in the MagLev device. Manipulation of the orientation of the levitating objects in space is achieved in two ways: (i) by rotating and/or translating the MagLev device while the objects are suspended in the paramagnetic solution between the magnets; (ii) by moving a small external magnet close to the levitating objects while keeping the device stationary. Unlike mechanical agitation or robotic selection, orienting using MagLev is possible for objects having a range of different physical characteristics (e.g., different shapes, sizes, and mechanical properties from hard polymers to gels and fluids). MagLev thus has the potential to be useful for sorting and positioning components in 3D space, orienting objects for assembly, constructing noncontact devices, and assembling objects composed of soft materials such as hydrogels, elastomers, and jammed granular media.

Shape Transformations of Lipid Bilayers Following Rapid Cholesterol Uptake.

High cholesterol levels in the blood increase the risk of atherosclerosis. A common explanation is that the cholesterol increase in the plasma membrane perturbs the shape and functions of cells by disrupting the cell signaling pathways and the formation of membrane rafts. In this work, we show that after enhanced transient uptake of cholesterol, mono-component lipid bilayers change their shape similarly to cell membranes in vivo. The bilayers either expel lipid protrusions or spread laterally as a result of the ensuing changes in their lipid density, the mechanical constraints imposed on them, and the properties of cyclodextrin used as a cholesterol donor. In light of the increasingly recognized link between membrane tension and cell behavior, we propose that the physical adaptation of the plasma membrane to cholesterol uptake may play a substantial role in the biological response.

Glycans pattern the phase behaviour of lipid membranes.

Hydrated networks of glycans (polysaccharides)--in the form of cell walls, periplasms or gel-like matrices--are ubiquitously present adjacent to cellular plasma membranes. Yet, despite their abundance, the function of glycans in the extracellular milieu is largely unknown. Here we show that the spatial configuration of glycans controls the phase behaviour of multiphase model lipid membranes: inhomogeneous glycan networks stabilize large lipid domains at the characteristic length scale of the network, whereas homogeneous networks suppress macroscopic lipid phase separation. We also find that glycan-patterned phase separation is thermally reversible--thus indicating that the effect is thermodynamic rather than kinetic--and that phase patterning probably results from a preferential interaction of glycans with ordered lipid phases. These findings have implications for membrane-mediated transport processes, potentially rationalize long-standing observations that differentiate the behaviour of native and model membranes and may indicate an intimate coupling between cellular lipidomes and glycomes.

Dynamics of giant vesicle assembly from thin lipid films.

MOTIVATION: Giant unilamellar vesicles (GUVs), cell-like synthetic micrometer size structures, assemble when thin lipid films are hydrated in aqueous solutions. Quantitative measurements of static yields and distribution of sizes of GUVs obtained from thin film hydration methods were recently reported. Dynamic data such as the time evolution of yields and distribution of sizes, however, is not known. Dynamic data can provide insights into the assembly pathway of GUVs and guidelines for choosing conditions to obtain populations with desired size distributions. APPROACH: We develop the 'stopped-time' technique to characterize the time evolution of the distribution of sizes and molar yields of populations of free-floating GUVs. We additionally capture high resolution time-lapse images of surface-attached GUV buds on the lipid films. We systematically study the dynamics of assembly of GUVs from three widely used thin film hydration methods, PAPYRUS (Paper-Abetted amPhiphile hYdRation in aqUeous Solutions), gentle hydration, and electroformation. FINDINGS: We find that the molar yield versus time curves of GUVs demonstrate a characteristic sigmoidal shape, with an initial yield, a transient, and then a steady state plateau for all three methods. The population of GUVs showed a right-skewed distribution of diameters. The variance of the distributions increased with time. The systems reached steady state within 120 min. We rationalize the dynamics using the thermodynamically motivated budding and merging (BNM) model. These results further the understanding of lipid dynamics and provide for the first-time practical parameters to tailor the production of GUVs of specific sizes for applications.

Insights into a clock's fidelity through vesicular encapsulation.

The single-celled cyanobacterium, Synechococcus elongatus , generates circadian rhythms with exceptional fidelity and synchrony despite their femtoliter volumes. Here, we explore the mechanistic aspects of this fidelity, by reconstituting the KaiABC post-translational oscillator (PTO) in cell-mimetic giant vesicles (GUVs) under well-defined conditions in vitro . PTO proteins were encapsulated with a coefficient of variation that closely matched protein variations observed in live cells. Using fluorescently labeled KaiB and confocal microscopy, we were able to measure circadian rhythms generated by thousands of encapsulated PTOs at the single-vesicle level for several days as a function of protein concentration and GUV size. We find that PTO fidelity decreased with decreasing levels of encapsulated PTO proteins and in smaller GUVs. We also observed that in encapsulated PTOs, a significant fraction of KaiB localized to GUV membranes like it does in cyanobacteria. A mathematical model that uses empirical bulk concentration and stoichiometry limitations suggests that cyanobacteria overcome challenges to fidelity by expressing high levels of PTO proteins along with the CikA and SasA proteins, which buffer stochastic variations in the levels of KaiA and KaiB, respectively. Further, the model suggests that the transcription-translation feedback loop (TTFL) contributes at most a small percentage to the overall fidelity of the cyanobacterial circadian clock under constant conditions but is essential for maintaining phase synchrony. Our results are the first experimental demonstration of populations of synthetic cells that can autonomously keep circadian time. Additionally, the approach of using bulk relationships to understand complex phenomena in cell-like systems could be useful for understanding other collective behavior important in biology, such as liquid-liquid phase separation.

Numb-associated kinases regulate sandfly-borne Toscana virus entry.

Sandfly-borne Toscana virus (TOSV) is an enveloped tri-segmented negative single-strand RNA Phlebovirus. It is an emerging virus predominantly endemic in southwestern Europe and Northern Africa. Although TOSV infection is typically asymptomatic or results in mild febrile disease, it is neurovirulent and ranks among the three most common causes of summer meningitis in certain regions. Despite this clinical significance, our understanding of the molecular aspects and host factors regulating phlebovirus infection is limited. This study characterized the early steps of TOSV infection. Our findings reveal that two members of the Numb-associated kinases family of Ser/Thr kinases, namely adaptor-associated kinase 1 (AAK1) and cyclin G-associated kinase (GAK), play a role in regulating the early stages of TOSV entry. FDA-approved inhibitors targeting these kinases demonstrated significant inhibition of TOSV infection. This study suggests that AAK1 and GAK represent druggable targets for inhibiting TOSV infection and, potentially, related Phleboviruses.

Spinal Anaesthesia for Urological Surgery: A Comparison of Isobaric Solutions of Levobupivacaine and Ropivacaine with Dexmedetomidine.

Background: Subarachnoid block is used in most of urological surgeries and finding the best possible drug has always been a challenge. Bupivacaine's pure enantiomers ropivacaine and levobupivacaine have lesser systemic toxicity. Isobaric solution has extra benefit of not affecting the intrathecal dispersion of drug. Dexmedetomidine when added intrathecally provides longer duration of analgesia and anaesthesia. Aim of this study is to compare onset, duration of the block with both the drugs along with their hemostability and postoperative analgesia. Methods: It is a Prospective Randomized Double-Blind Study. It includes 68 patients undergoing urological procedures under subarachnoid block. Group LD: Patients will receive 3.5 ml of Isobaric Levobupivacaine 0.5% + Dexmedetomidine 10 µg (0.1ml) Group RD: will receive 3.5ml of Isobaric Ropivacaine 0.5% + Dexmedetomidine 10 µg (0.1ml). Results: Time taken for onset of sensory and motor block is significantly more in ropivacaine while duration of block is more in levobupivacaine. Conclusions: Addition of Dexmedetomidine to Isobaric Levobupivacaine significantly prolongs the duration of analgesia and anaesthesia compared to Ropivacaine and maintains stable hemodynamics. Ropivacaine is a suitable drug for day care whilst levobupivacaine is an excellent agent for longer surgeries. Dexmedetomidine is an effective non-opioid adjuvant which improves effectiveness of block without increasing the risk of side effects.

Son of sevenless 1 (SOS1), the RasGEF, interacts with ERα and STAT3 during embryo implantation.

Estrogen accounts for several biological processes in the body; embryo implantation and pregnancy being one of the vital events. This manuscript aims to unearth the nuclear role of Son of sevenless1 (SOS1), its interaction with estrogen receptor alpha (ERα), and signal transducer and activator of transcription 3 (STAT3) in the uterine nucleus during embryo implantation. SOS1, a critical cytoplasmic linker between receptor tyrosine kinase and rat sarcoma virus signaling, translocates into the nucleus via its bipartite nuclear localization signal (NLS) during the 'window of implantation' in pregnant mice. SOS1 associates with chromatin, interacts with histones, and shows intrinsic histone acetyltransferase (HAT) activity specifically acetylating lysine 16 (K16) residue of histone H4. SOS1 is a coactivator of STAT3 and a co-repressor of ERα. SOS1 creates a partial mesenchymal-epithelial transition by acting as a transcriptional modulator. Finally, our phylogenetic tree reveals that the two bipartite NLS surface in reptiles and the second acetyl coenzymeA (CoA) (RDNGPG) important for HAT activity emerges in mammals. Thus, SOS1 has evolved into a moonlighting protein, the special class of multi-tasking proteins, by virtue of its newly identified nuclear functions in addition to its previously known cytoplasmic function.

Effect of Subanaesthetic Dose of Ketamine on Pneumoperitoneal Response and Clinical Recovery in Patients Undergoing Laparoscopy.

OBJECTIVE: Although suppression of intraperitoneal gas insufflation response is possible with a higher dose of opioids, sedatives, and inha- lational agents, delayed emergence and poor clinical recovery are still a matter of concern. Here our primary aim was to assess the quality of recovery and the secondary aim includes postinsufflation response, postoperative pain intensity, total opioid requirement, and looking for adverse effects, if any. METHODS: This prospective randomized double-blinded controlled study was conducted among 75 American Society of Anesthesiologist physical status I and II patients scheduled for laparoscopic surgeries under general anaesthesia. Group 1 received injection tramadol 1 mg kg-1 iv-1 5 minutes after intubation. Similarly, groups 2 and 3 received 0.25 mg kg-1 and 0.5 mg kg-1 injection of ketamine iv, respectively. Intraperitoneal insufflation response was observed from the beginning of insufflation till 15 minutes. Clinical recovery was measured in terms of vigilance, cognition, orientation, and comfort. Postoperative pain intensity was assessed at varying movement activities using numerical rating scale pain score and with the total opioid requirement. The collected data were analyzed using three-way ANOVA. RESULTS: Groups 1 and 2 had a fair clinical recovery. Postoperative pain intensity was least in group 2, and the postinsufflation mean arterial pressure was higher in groups 1 and 3. A total of 32% of participants had delirium in group 3. CONCLUSIONS: Clinical recovery and perioperative analgesia were better in ketamine group (0.25 mg kg-1) without any perturbations in intra- operative pneumoperitoneal response. Hence it can be considered an optimal adjuvant in laparoscopic surgeries.

Colloid science: non-spherical bubbles.

Surface tension gives gas bubbles their perfect spherical shape by minimizing the surface area for a given volume. Here we show that gas bubbles and liquid drops can exist in stable, non-spherical shapes if the surface is covered, or 'armoured', with a close-packed monolayer of particles. When two spherical armoured bubbles are fused, jamming of the particles on the interface supports the unequal stresses that are necessary to stabilize a non-spherical shape.

Nanoscale Curvature Promotes High Yield Spontaneous Formation of Cell-Mimetic Giant Vesicles on Nanocellulose Paper.

To date, techniques for the assembly of phospholipid films into cell-like giant unilamellar vesicles (GUVs) use planar surfaces and require the application of electric fields or dissolved molecules to obtain adequate yields. Here, we present the use of nanocellulose paper, which are surfaces composed of entangled cylindrical nanofibers, to promote the facile and high yield assembly of GUVs. Use of nanocellulose paper results in up to a 100 000-fold reduction in costs while increasing yields compared to extant surface-assisted assembly techniques. Quantitative measurements of yields and the distributions of sizes using large data set confocal microscopy illuminates the mechanism of assembly. We present a thermodynamic "budding and merging", BNM, model that offers a unified explanation for the differences in the yields and sizes of GUVs obtained from surfaces of varying geometry and chemistry. The BNM model considers the change in free energy due to budding by balancing the elastic, adhesion, and edge energies of a section of a surface-attached membrane that transitions into a surface-attached spherical bud. The model reveals that the formation of GUVs is spontaneous on hydrophilic surfaces consisting of entangled cylindrical nanofibers with dimensions similar to nanocellulose fibers. This work advances understanding of the effects of surface properties on the assembly of GUVs. It also addresses practical barriers that currently impede the promising use of GUVs as vehicles for the delivery of drugs, for the manufacturing of synthetic cells, and for the assembly of artificial tissues at scale.

Derivation of iPSC lines from two patients with familial Alzheimer's disease from India.

The current prevalence of diagnosable dementia in India is 1% of people over 60 years (~3.7 million people), but is estimated to increase significantly, as ~15% world's aged population (>65 years) would be resident here by 2020 (Shah et al., 2016). While several mutations that pose a familial risk have been identified, the ethnic background may influence disease susceptibility, clinical presentation and treatment response. In this study, we report a detailed characterization of two representative HiPSC lines from a well-characterized dementia cohort from India. Availability of these lines, and associated molecular and clinical information, would be useful in the detailed exploration of the genomic contribution(s) to AD.

Emulsification efficacy of Quillaja saponins at very low concentration: Model development and role of alcohols.

The present study aims at quantifying interfacial coverage of a biosurfactant (Quillaja saponins) and understanding the impact of flavor and fragrance alcohols on emulsification efficacy of the biosurfactant in a surfactant-oil-matrix system. Emulsions were prepared using limonene, alkanes (C8, C12, and C16) or limonene ̶ alcohol (linalool and C6C10 alcohols) mixtures at different ratios as oil phase stabilized by Quillaja saponins at very low concentrations (0.005-0.05% w/w). Droplet size was measured and size distributions were numerized to determine surface and volume average droplet diameters of bimodal emulsions. Using a model developed in the present study, Quillaja saponins showed an interfacial coverage of 5.0×106cm2/g and a head surface of 1.37nm2 with a lay-on configuration at interface. The model proved to discriminate between surface active (alcohols) and non-active (alkanes) compounds. The apparent interfacial coverage of saponins increased linearly with increasing alcohol concentration. The type of alcohol (terpene alcohol vs. medium chain alcohols) and alcohol chain length (C6C10) showed little impact on emulsification efficacy of Quillaja saponins. The molar ratio of heptanol to saponin at interface increased from 0 to 8.6 corresponding to 0-30% w/w heptanol in limonene. This study revealed that the distribution of alcohol at interface was mainly driven by partitioning in the surfactant-oil-matrix system. The practical implication of the present study is to enhance emulsification efficacy of Quillaja Saponins at very low concentration by incorporating surface active compounds, i.e. flavor or fragrance alcohols.

Lipid Bilayers Are Long-Lived on Solvent Cleaned Plasma-Oxidized poly(dimethyl)siloxane (ox-PDMS).

Although it is well known that phospholipids self-assemble on hydrophilic plasma-oxidized PMDS surfaces (ox-PDMS) to form cell membrane mimetic bilayers, the temporal stability of phospholipid membranes on these surfaces is unknown. Here we report that phospholipid bilayers remain stable on solvent-cleaned ox-PDMS for at least 132 hours after preparation. Absent solvent cleaning, the bilayers were stable for only 36 hours. We characterized the phospholipid bilayers, i) through quantitative comparative analysis of the fluorescence intensity of phospholipid bilayers on ox-PDMS and phospholipid monolayers on native PDMS and, ii) through measurements of the diffusive mobility of the lipids through fluorescence recovery after photobleaching (FRAP). The fluorescence intensity of the phospholipid layer remained consistent with that of a bilayer for 132 hours. The evolution of the diffusive mobility of the phospholipids in the bilayer on ox-PDMS over time was similar to lipids in control bilayers prepared on glass surfaces. Solvent cleaning was essential for the long-term stability of the bilayers on ox-PDMS. Without cleaning in acetone and isopropanol, phospholipid bilayers prepared on ox-PDMS surfaces peeled off in large patches within 36 hours. Importantly, we find that phospholipid bilayers supported on solvent-cleaned ox-PDMS were indistinguishable from phospholipid bilayers supported on glass for at least 36 hours after preparation. Our results provide a link between the two common surfaces used to prepare in vitro biomimetic phospholipid membranes-i) glass surfaces used predominantly in fundamental biophysical experiments, for which there is abundant physicochemical information, with ii) ox-PDMS, the dominant material used in practical, applications-oriented systems to build micro-devices, topographically-patterned surfaces, and biosensors where there is a dearth of information.