Protein-Rich Rafts in Hybrid Polymer/Lipid Giant Unilamellar Vesicles.

Considerable attention has been dedicated to lipid rafts due to their importance in numerous cell functions such as membrane trafficking, polarization, and signaling. Next to studies in living cells, artificial micrometer-sized vesicles with a minimal set of components are established as a major tool to understand the phase separation dynamics and their intimate interplay with membrane proteins. In parallel, mixtures of phospholipids and certain amphiphilic polymers simultaneously offer an interface for proteins and mimic this segregation behavior, presenting a tangible synthetic alternative for fundamental studies and bottom-up design of cellular mimics. However, the simultaneous insertion of complex and sensitive membrane proteins is experimentally challenging and thus far has been largely limited to natural lipids. Here, we present the co-reconstitution of the proton pump bo3 oxidase and the proton consumer ATP synthase in hybrid polymer/lipid giant unilamellar vesicles (GUVs) via fusion/electroformation. Variations of the current method allow for tailored reconstitution protocols and control of the vesicle size. In particular, mixing of protein-free and protein-functionalized nanosized vesicles in the electroformation film results in larger GUVs, while separate reconstitution of the respiratory enzymes enables higher ATP synthesis rates. Furthermore, protein labeling provides a synthetic mechanism for phase separation and protein sequestration, mimicking lipid- and protein-mediated domain formation in nature. The latter means opens further possibilities for re-enacting phenomena like supercomplex assembly or symmetry breaking and enriches the toolbox of bottom-up synthetic biology.

Light-Driven ATP Regeneration in Diblock/Grafted Hybrid Vesicles.

Light-driven ATP regeneration systems combining ATP synthase and bacteriorhodopsin have been proposed as an energy supply in the field of synthetic biology. Energy is required to power biochemical reactions within artificially created reaction compartments like protocells, which are typically based on either lipid or polymer membranes. The insertion of membrane proteins into different hybrid membranes is delicate, and studies comparing these systems with liposomes are needed. Here we present a detailed study of membrane protein functionality in different hybrid compartments made of graft polymer PDMS-g-PEO and diblock copolymer PBd-PEO. Activity of more than 90 % in lipid/polymer-based hybrid vesicles could prove an excellent biocompatibility. A significant enhancement of long-term stability (80 % remaining activity after 42 days) could be demonstrated in polymer/polymer-based hybrids.

Artificial Organelles for Energy Regeneration.

One of the critical steps in sustaining life-mimicking processes in synthetic cells is energy, i.e., adenosine triphosphate (ATP) regeneration. Previous studies have shown that the simple addition of ATP or ATP regeneration systems, which do not regenerate ATP directly from ADP and Pi , have no or only limited success due to accumulation of ATP hydrolysis products. In general, ATP regeneration can be achieved by converting light or chemical energy into ATP, which may also involve redox transformations of cofactors. The present contribution provides an overview of the existing ATP regeneration strategies and the related nicotinamide adenine dinucleotide (NAD+ ) redox cycling, with a focus on compartmentalized systems. Special attention is being paid to those approaches where so-called artificial organelles are developed. They comprise a semipermeable membrane functionalized by biological or man-made components and employ external energy in the form of light or nutrients in order to generate a transmembrane proton gradient, which is further utilized for ATP synthesis.

Out-of-equilibrium microcompartments for the bottom-up integration of metabolic functions.

Self-sustained metabolic pathways in microcompartments are the corner-stone for living systems. From a technological viewpoint, such pathways are a mandatory prerequisite for the reliable design of artificial cells functioning out-of-equilibrium. Here we develop a microfluidic platform for the miniaturization and analysis of metabolic pathways in man-made microcompartments formed of water-in-oil droplets. In a modular approach, we integrate in the microcompartments a nicotinamide adenine dinucleotide (NAD)-dependent enzymatic reaction and a NAD-regeneration module as a minimal metabolism. We show that the microcompartments sustain a metabolically active state until the substrate is fully consumed. Reversibly, the external addition of the substrate reboots the metabolic activity of the microcompartments back to an active state. We therefore control the metabolic state of thousands of independent monodisperse microcompartments, a step of relevance for the construction of large populations of metabolically active artificial cells.

Toward Artificial Mitochondrion: Mimicking Oxidative Phosphorylation in Polymer and Hybrid Membranes.

For energy supply to biomimetic constructs, a complex chemical energy-driven ATP-generating artificial system was built. The system was assembled with bottom-up detergent-mediated reconstitution of an ATP synthase and a terminal oxidase into two types of novel nanocontainers, built from either graft copolymer membranes or from hybrid graft copolymer/lipid membranes. The versatility and biocompatibility of the proposed nanocontainers was demonstrated through convenient system assembly and through high retained activity of both membrane-embedded enzymes. In the future, the nanocontainers might be used as a platform for the functional reconstitution of other complex membrane proteins and could considerably expedite the design of nanoreactors, biosensors, and artificial organelles.

Perfil orofacial de crianças respiradoras orais pré adenoidectomia e/ou amidalectomia / Orofacial profile of mouth breathing children previous to adenoidectomy and/or tonsillectomy / Perfil orofacial en niños con respiración oral antes de una adenoidectomía y/o amigdalectomía

As obstruções das vias aéreas superiores, como tonsilas hipertrofiadas, são causas da respiração oral. Objetivo: traçar o perfil miofuncional orofacial de crianças respiradoras orais pré-adenoidectomia e/ou amidalectomia. Trata-se de um estudo descritivo de caráter quantitativo de levantamento de dados. As avaliações de motricidade orofacial basearam-se no protocolo MBGR. Resultados: foram avaliadas 32 crianças, com média de idade de 8,9 anos. Destas, 34,4% eram do sexo feminino e 65,6%, do sexo masculino; prevaleceram o aleitamento natural e o hábito oral. As principais queixas foram de respiração oral e ronco. A maioria foi encaminhada para a realização de adenoamigdalectomia. A postura corporal esteve normal. Prevaleceram lábios entreabertos e língua no assoalho bucal, má oclusão Classe II de Angle, presença de tonsilas hipertrofiadas. O ronco foi descrito em 100% da amostra. A deglutição atípica prevaleceu em 77,4%. A articulação apresentou-se predominantemente normal, as alterações articulatórias presentes foram observadas no sexo masculino. A postura de lábios entreabertos apresentou relação com a deglutição atípica. Conclusão: os dados levantados não diferem da literatura com relação à respiração oral obstrutiva. Abre-se espaço para pesquisas que abranjam o pré e pós-operatório, e que propiciem um acompanhamento longitudinal das crianças.

The obstruction of the upper airways, such as hypertrophic tonsils, is the main cause of mouth breathing. Objective: to trace the orofacial myofunctional profile of mouth-breathing children previous to adenoidectomy and/or tonsillectomy. This is a descriptive quantitative study for data collection. Evaluations of orofacial motor function were based on the MBGR protocol. Results: 32 children with an age average of 8.9 years were evaluated; 34.4% of them were female and 65.6% male; breastfeeding and oral habits prevailed. The main complaints were oral breathing and snoring. Most were referred for adenotonsillectomy. The body posture was normal in 68.8% of the cases. There was a prevalence of parted lips and tongue in the mouth floor, Angle Class II malocclusion, besides hypertrophic tonsils. Snoring was described in 100% of the sample. Atypical swallowing was present in 77.4% of the sample. The articulation was predominantly normal, and articular changes were observed in males. Parted lips were related to atypical swallowing. Conclusion: the collected data did not differ from the literature regarding obstructive oral breathing. There is an open space for new research that embraces both pre- and postoperatory moments, providing a longitudinal monitoring of children.

Las obstrucciones de las vías respiratorias superiores, tales como las amígdalas hipertrofiadas, son la causa de la respiración oral. Objetivo: Trazar el perfil miofuncional orofacial de niños antes de la adenoidectomía y/o amigdalectomía. Se trata de un estudio descriptivo y cuantitativo para la recopilación de datos. Las evaluaciones de la función motora orofacial se basaran en el protocolo MBGR. Resultados: Se evaluaron 32 niños con un pormedio de edad de 8,9 años. De estos, el 34,4% eran mujeres y 65,6%, hombres; han prevalecido la lactancia materna y el hábito oral. Las principales quejas eran la respiración oral y ronquidos. La mayoría fue remitida para la realización de adenoamigdalectomía. La postura del cuerpo ha prevalecido normal. Hubo prevalencia de labios entreabiertos y de lengua en el piso de la boca, maloclusión Clase II de Angle y la presencia de amígdalas hipertrofiadas. El ronquido fue descrito en el 100% de los casos. La función masticatoria era normal. La deglución atípica estaba presente en 77,4%. La articulación se presentó predominantemente normal, las alteraciones articulatorias presentes fueron observadas en el sexo masculino. Los labios entreabiertos presentaran relación con la deglución atípica. Conclusión: los datos levantados no difieren de la literatura acerca de la respiración oral de etiología obstructiva. Se abre espacio para investigaciones que cubran los períodos pre y postoperatorio y propicien un seguimiento longitudinal de los niños.