Evidence of an upper ionospheric electric field perturbation correlated with a gamma ray burst.

Earth's atmosphere, whose ionization stability plays a fundamental role for the evolution and endurance of life, is exposed to the effect of cosmic explosions producing high energy Gamma-ray-bursts. Being able to abruptly increase the atmospheric ionization, they might deplete stratospheric ozone on a global scale. During the last decades, an average of more than one Gamma-ray-burst per day were recorded. Nevertheless, measurable effects on the ionosphere were rarely observed, in any case on its bottom-side (from about 60 km up to about 350 km of altitude). Here, we report evidence of an intense top-side (about 500 km) ionospheric perturbation induced by significant sudden ionospheric disturbance, and a large variation of the ionospheric electric field at 500 km, which are both correlated with the October 9, 2022 Gamma-ray-burst (GRB221009A).

In situ architecture of the ER-mitochondria encounter structure.

The endoplasmic reticulum and mitochondria are main hubs of eukaryotic membrane biogenesis that rely on lipid exchange via membrane contact sites1-3, but the underpinning mechanisms remain poorly understood. In yeast, tethering and lipid transfer between the two organelles is mediated by the endoplasmic reticulum-mitochondria encounter structure (ERMES), a four-subunit complex of unresolved stoichiometry and architecture4-6. Here we determined the molecular organization of ERMES within Saccharomyces cerevisiae cells using integrative structural biology by combining quantitative live imaging, cryo-correlative microscopy, subtomogram averaging and molecular modelling. We found that ERMES assembles into approximately 25 discrete bridge-like complexes distributed irregularly across a contact site. Each bridge consists of three synaptotagmin-like mitochondrial lipid binding protein domains oriented in a zig-zag arrangement. Our molecular model of ERMES reveals a pathway for lipids. These findings resolve the in situ supramolecular architecture of a major inter-organelle lipid transfer machinery and provide a basis for the mechanistic understanding of lipid fluxes in eukaryotic cells.

DNA Self-Assembly of Single Molecules with Deterministic Position and Orientation.

An ideal nanofabrication method should allow the organization of nanoparticles and molecules with nanometric positional precision, stoichiometric control, and well-defined orientation. The DNA origami technique has evolved into a highly versatile bottom-up nanofabrication methodology that fulfils almost all of these features. It enables the nanometric positioning of molecules and nanoparticles with stoichiometric control, and even the orientation of asymmetrical nanoparticles along predefined directions. However, orienting individual molecules has been a standing challenge. Here, we show how single molecules, namely, Cy5 and Cy3 fluorophores, can be incorporated in a DNA origami with controlled orientation by doubly linking them to oligonucleotide strands that are hybridized while leaving unpaired bases in the scaffold. Increasing the number of bases unpaired induces a stretching of the fluorophore linkers, reducing its mobility freedom, and leaves more space for the fluorophore to accommodate and find different sites for interaction with the DNA. Particularly, we explore the effects of leaving 0, 2, 4, 6, and 8 bases unpaired and find extreme orientations for 0 and 8 unpaired bases, corresponding to the molecules being perpendicular and parallel to the DNA double-helix, respectively. We foresee that these results will expand the application field of DNA origami toward the fabrication of nanodevices involving a wide range of orientation-dependent molecular interactions, such as energy transfer, intermolecular electron transport, catalysis, exciton delocalization, or the electromagnetic coupling of a molecule to specific resonant nanoantenna modes.

PEOT/PBT Polymeric Pastes to Fabricate Additive Manufactured Scaffolds for Tissue Engineering.

The advantages of additive manufactured scaffolds, as custom-shaped structures with a completely interconnected and accessible pore network from the micro- to the macroscale, are nowadays well established in tissue engineering. Pore volume and architecture can be designed in a controlled fashion, resulting in a modulation of scaffold's mechanical properties and in an optimal nutrient perfusion determinant for cell survival. However, the success of an engineered tissue architecture is often linked to its surface properties as well. The aim of this study was to create a family of polymeric pastes comprised of poly(ethylene oxide therephthalate)/poly(butylene terephthalate) (PEOT/PBT) microspheres and of a second biocompatible polymeric phase acting as a binder. By combining microspheres with additive manufacturing technologies, we produced 3D scaffolds possessing a tailorable surface roughness, which resulted in improved cell adhesion and increased metabolic activity. Furthermore, these scaffolds may offer the potential to act as drug delivery systems to steer tissue regeneration.

Deactivation blocks proton pathways in the mitochondrial complex I.

Cellular respiration is powered by membrane-bound redox enzymes that convert chemical energy into an electrochemical proton gradient and drive the energy metabolism. By combining large-scale classical and quantum mechanical simulations with cryo-electron microscopy data, we resolve here molecular details of conformational changes linked to proton pumping in the mammalian complex I. Our data suggest that complex I deactivation blocks water-mediated proton transfer between a membrane-bound quinone site and proton-pumping modules, decoupling the energy-transduction machinery. We identify a putative gating region at the interface between membrane domain subunits ND1 and ND3/ND4L/ND6 that modulates the proton transfer by conformational changes in transmembrane helices and bulky residues. The region is perturbed by mutations linked to human mitochondrial disorders and is suggested to also undergo conformational changes during catalysis of simpler complex I variants that lack the "active"-to-"deactive" transition. Our findings suggest that conformational changes in transmembrane helices modulate the proton transfer dynamics by wetting/dewetting transitions and provide important functional insight into the mammalian respiratory complex I.

Molecular strain in the active/deactive-transition modulates domain coupling in respiratory complex I.

Complex I functions as a primary redox-driven proton pump in aerobic respiratory chains, establishing a proton motive force that powers ATP synthesis and active transport. Recent cryo-electron microscopy (cryo-EM) experiments have resolved the mammalian complex I in the biomedically relevant active (A) and deactive (D) states (Zhu et al., 2016; Fiedorczuk et al., 2016; Agip et al., 2018 [1-3]) that could regulate enzyme turnover, but it still remains unclear how the conformational state and activity are linked. We show here how global motion along the A/D transition accumulates molecular strain at specific coupling regions important for both redox chemistry and proton pumping. Our data suggest that the A/D motion modulates force propagation pathways between the substrate-binding site and the proton pumping machinery that could alter electrostatic and conformational coupling across large distances. Our findings provide a molecular basis to understand how global protein dynamics can modulate the biological activity of large molecular complexes.

Inconsistency in Graft Outcome of Bilayered Bioresorbable Supramolecular Arterial Scaffolds in Rats.

There is a continuous search for the ideal bioresorbable material to develop scaffolds for in situ vascular tissue engineering. As these scaffolds are exposed to the harsh hemodynamic environment during the entire transformation process from scaffold to neotissue, it is of crucial importance to maintain mechanical integrity and stability at all times. Bilayered scaffolds made of supramolecular polycarbonate-ester-bisurea were manufactured using dual electrospinning. These scaffolds contained a porous inner layer to allow for cellular infiltration and a dense outer layer to provide strength. Scaffolds (n = 21) were implanted as an interposition graft into the abdominal aorta of male Lewis rats and explanted after 1, 3, and 5 months in vivo to assess mechanical functionality and neotissue formation upon scaffold resorption. Results demonstrated conflicting graft outcomes despite homogeneity in the experimental group and scaffold production. Most grafts exhibited adverse remodeling, resulting in aneurysmal dilatation and calcification. However, a few grafts did not demonstrate such features, but instead were characterized by graft extension and smooth muscle cell proliferation in the absence of endothelium, while remaining patent throughout the study. We conclude that it remains extremely difficult to anticipate graft development and performance in vivo. Next to rational mechanical design and good performance in vitro, a thorough understanding of the mechanobiological mechanisms governing scaffold-driven arterial regeneration as well as potential influences of surgical procedures is warranted to further optimize scaffold designs. Careful analysis of the differences between preclinical successes and failures, as is done in this study, may provide initial handles for scaffold optimization and standardized surgical procedures to improve graft performance in vivo. Impact statement In situ vascular tissue engineering using cell-free bioresorbable scaffolds is investigated as an off-the-shelf option to grow small caliber arteries inside the body. In this study, we developed a bilayered electrospun supramolecular scaffold with a dense outer layer to provide mechanical integrity and a porous inner layer for cell recruitment and tissue formation. Despite homogenous scaffold properties and mechanical performance in vitro, in vivo testing as rat aorta interposition grafts revealed distinct graft outcomes, ranging from aneurysms to functional arteries. Careful analysis of this variability provided valuable insights into materials-driven in situ artery formation relevant for scaffold design and implantation procedures.

Fe-chitosan complexes for oxidative degradation of emerging contaminants in water: Structure, activity, and reaction mechanism.

Versatile and ecofriendly methods to perform oxidations at near-neutral pH are of crucial importance for processes aimed at purifying water. Chitosan, a deacetylated form of chitin, is a promising starting material owing to its biocompatibility and ability to form stable films and complexes with metals. Here, we report a novel chitosan-based organometallic complex that was tested both as homogeneous and heterogeneous catalyst in the degradation of contaminants of emerging concern in water. The stoichiometry of the complex was experimentally verified with different metals, namely, Cu(II), Fe(III), Fe(II), Co(II), Pd(II), and Mn(II), and we identified the chitosan-Fe(III) complex as the most efficient catalyst. This complex effectively degraded phenol, triclosan, and 3-chlorophenol in the presence of hydrogen peroxide. A putative ferryl-mediated reaction mechanism is proposed based on experimental data, density functional theory calculations, and kinetic modeling. Finally, a film of the chitosan-Fe(III) complex was synthesized and proven a promising supported heterogeneous catalyst for water purification.

Structure of inhibitor-bound mammalian complex I.

Respiratory complex I (NADH:ubiquinone oxidoreductase) captures the free energy from oxidising NADH and reducing ubiquinone to drive protons across the mitochondrial inner membrane and power oxidative phosphorylation. Recent cryo-EM analyses have produced near-complete models of the mammalian complex, but leave the molecular principles of its long-range energy coupling mechanism open to debate. Here, we describe the 3.0-Å resolution cryo-EM structure of complex I from mouse heart mitochondria with a substrate-like inhibitor, piericidin A, bound in the ubiquinone-binding active site. We combine our structural analyses with both functional and computational studies to demonstrate competitive inhibitor binding poses and provide evidence that two inhibitor molecules bind end-to-end in the long substrate binding channel. Our findings reveal information about the mechanisms of inhibition and substrate reduction that are central for understanding the principles of energy transduction in mammalian complex I.

Water-Gated Proton Transfer Dynamics in Respiratory Complex I.

The respiratory complex I transduces redox energy into an electrochemical proton gradient in aerobic respiratory chains, powering energy-requiring processes in the cell. However, despite recently resolved molecular structures, the mechanism of this gigantic enzyme remains poorly understood. By combining large-scale quantum and classical simulations with site-directed mutagenesis and biophysical experiments, we show here how the conformational state of buried ion-pairs and water molecules control the protonation dynamics in the membrane domain of complex I and establish evolutionary conserved long-range coupling elements. We suggest that an electrostatic wave propagates in forward and reverse directions across the 200 Å long membrane domain during enzyme turnover, without significant dissipation of energy. Our findings demonstrate molecular principles that enable efficient long-range proton-electron coupling (PCET) and how perturbation of this PCET machinery may lead to development of mitochondrial disease.

In Situ Remodeling Overrules Bioinspired Scaffold Architecture of Supramolecular Elastomeric Tissue-Engineered Heart Valves.

In situ tissue engineering that uses resorbable synthetic heart valve scaffolds is an affordable and practical approach for heart valve replacement; therefore, it is attractive for clinical use. This study showed no consistent collagen organization in the predefined direction of electrospun scaffolds made from a resorbable supramolecular elastomer with random or circumferentially aligned fibers, after 12 months of implantation in sheep. These unexpected findings and the observed intervalvular variability highlight the need for a mechanistic understanding of the long-term in situ remodeling processes in large animal models to improve predictability of outcome toward robust and safe clinical application.

How cardiolipin modulates the dynamics of respiratory complex I.

Cardiolipin modulates the activity of membrane-bound respiratory enzymes that catalyze biological energy transduction. The respiratory complex I functions as the primary redox-driven proton pump in mitochondrial and bacterial respiratory chains, and its activity is strongly enhanced by cardiolipin. However, despite recent advances in the structural biology of complex I, cardiolipin-specific interaction mechanisms currently remain unknown. On the basis of millisecond molecular simulations, we suggest that cardiolipin binds to proton-pumping subunits of complex I and induces global conformational changes that modulate the accessibility of the quinone substrate to the enzyme. Our findings provide key information on the coupling between complex I dynamics and activity and suggest how biological membranes modulate the structure and activity of proteins.

3D-printed bioactive scaffolds from nanosilicates and PEOT/PBT for bone tissue engineering.

Additive manufacturing (AM) has shown promise in designing 3D scaffold for regenerative medicine. However, many synthetic biomaterials used for AM are bioinert. Here, we report synthesis of bioactive nanocomposites from a poly(ethylene oxide terephthalate) (PEOT)/poly(butylene terephthalate) (PBT) (PEOT/PBT) copolymer and 2D nanosilicates for fabricating 3D scaffolds for bone tissue engineering. PEOT/PBT have been shown to support calcification and bone bonding ability in vivo, while 2D nanosilicates induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) in absence of osteoinductive agents. The effect of nanosilicates addition to PEOT/PBT on structural, mechanical and biological properties is investigated. Specifically, the addition of nanosilicate to PEOT/PBT improves the stability of nanocomposites in physiological conditions, as nanosilicate suppressed the degradation rate of copolymer. However, no significant increase in the mechanical stiffness of scaffold due to the addition of nanosilicates is observed. The addition of nanosilicates to PEOT/PBT improves the bioactive properties of AM nanocomposites as demonstrated in vitro. hMSCs readily proliferated on the scaffolds containing nanosilicates and resulted in significant upregulation of osteo-related proteins and production of mineralized matrix. The synergistic ability of nanosilicates and PEOT/PBT can be utilized for designing bioactive scaffolds for bone tissue engineering.

Redox-coupled quinone dynamics in the respiratory complex I.

Complex I couples the free energy released from quinone (Q) reduction to pump protons across the biological membrane in the respiratory chains of mitochondria and many bacteria. The Q reduction site is separated by a large distance from the proton-pumping membrane domain. To address the molecular mechanism of this long-range proton-electron coupling, we perform here full atomistic molecular dynamics simulations, free energy calculations, and continuum electrostatics calculations on complex I from Thermus thermophilus We show that the dynamics of Q is redox-state-dependent, and that quinol, QH2, moves out of its reduction site and into a site in the Q tunnel that is occupied by a Q analog in a crystal structure of Yarrowia lipolytica We also identify a second Q-binding site near the opening of the Q tunnel in the membrane domain, where the Q headgroup forms strong interactions with a cluster of aromatic and charged residues, while the Q tail resides in the lipid membrane. We estimate the effective diffusion coefficient of Q in the tunnel, and in turn the characteristic time for Q to reach the active site and for QH2 to escape to the membrane. Our simulations show that Q moves along the Q tunnel in a redox-state-dependent manner, with distinct binding sites formed by conserved residue clusters. The motion of Q to these binding sites is proposed to be coupled to the proton-pumping machinery in complex I.

Dual Electrospun Supramolecular Polymer Systems for Selective Cell Migration.

Dual electrospinning can be used to make multifunctional scaffolds for regenerative medicine applications. Here, two supramolecular polymers with different material properties are electrospun simultaneously to create a multifibrous mesh. Bisurea (BU)-based polycaprolactone, an elastomer providing strength to the mesh, and ureido-pyrimidinone (UPy) modified poly(ethylene glycol) (PEG), a hydrogelator, introducing the capacity to deliver compounds upon swelling. The dual spun scaffolds are modularly tuned by mixing UPyPEG hydrogelators with different polymer lengths, to control swelling of the hydrogel fiber, while maintaining the mechanical properties of the scaffold. Stromal cell derived factor 1 alpha (SDF1α) peptides are embedded in the UPyPEG fibers. The swelling and erosion of UPyPEG increase void spaces and released the SDF1α peptide. The functionalized scaffolds demonstrate preferential lymphocyte recruitment proposed to be created by a gradient formed by the released SDF1α peptide. This delivery approach offers the potential to develop multifibrous scaffolds with various functions.

How inter-subunit contacts in the membrane domain of complex I affect proton transfer energetics.

The respiratory complex I is a redox-driven proton pump that employs the free energy released from quinone reduction to pump protons across its complete ca. 200 Šwide membrane domain. Despite recently resolved structures and molecular simulations, the exact mechanism for the proton transport process remains unclear. Here we combine large-scale molecular simulations with quantum chemical density functional theory (DFT) models to study how contacts between neighboring antiporter-like subunits in the membrane domain of complex I affect the proton transfer energetics. Our combined results suggest that opening of conserved Lys/Glu ion pairs within each antiporter-like subunit modulates the barrier for the lateral proton transfer reactions. Our work provides a mechanistic suggestion for key coupling effects in the long-range force propagation process of complex I.

Global collective motions in the mammalian and bacterial respiratory complex I.

The respiratory complex I is an enzyme responsible for the conversion of chemical energy into an electrochemical proton motive force across the membrane. Despite extensive studies, the mechanism by which the activity of this enormous, ca. 1 MDa, redox-coupled proton pump is regulated still remains unclear. Recent structural studies (Zhu et al., Nature 2016; Fiedorczuk et al., Nature 2016) resolved complex I in different conformations connected to the active-to-deactive (A/D) transition that regulate complex I activity in several species. Based on anisotropic network models (ANM) and principal component analysis (PCA), we identify here transitions between experimentally resolved structures of the mammalian complex I as low-frequency collective motions of the enzyme, highlighting similarities and differences between the bacterial and mammalian enzymes. Despite the reduced complexity of the smaller bacterial enzyme, our results suggest that the global dynamics of complex I is overall conserved. We further probe how the supernumerary subunits could be involved in the modulation of the A/D-transition, and show that in particular the 42 kDa and B13 subunits affect the global motions of the mammalian enzyme.

Correlating kinetic and structural data on ubiquinone binding and reduction by respiratory complex I.

Respiratory complex I (NADH:ubiquinone oxidoreductase), one of the largest membrane-bound enzymes in mammalian cells, powers ATP synthesis by using the energy from electron transfer from NADH to ubiquinone-10 to drive protons across the energy-transducing mitochondrial inner membrane. Ubiquinone-10 is extremely hydrophobic, but in complex I the binding site for its redox-active quinone headgroup is ∼20 Šabove the membrane surface. Structural data suggest it accesses the site by a narrow channel, long enough to accommodate almost all of its ∼50-Šisoprenoid chain. However, how ubiquinone/ubiquinol exchange occurs on catalytically relevant timescales, and whether binding/dissociation events are involved in coupling electron transfer to proton translocation, are unknown. Here, we use proteoliposomes containing complex I, together with a quinol oxidase, to determine the kinetics of complex I catalysis with ubiquinones of varying isoprenoid chain length, from 1 to 10 units. We interpret our results using structural data, which show the hydrophobic channel is interrupted by a highly charged region at isoprenoids 4-7. We demonstrate that ubiquinol-10 dissociation is not rate determining and deduce that ubiquinone-10 has both the highest binding affinity and the fastest binding rate. We propose that the charged region and chain directionality assist product dissociation, and that isoprenoid stepping ensures short transit times. These properties of the channel do not benefit the exhange of short-chain quinones, for which product dissociation may become rate limiting. Thus, we discuss how the long channel does not hinder catalysis under physiological conditions and the possible roles of ubiquinone/ubiquinol binding/dissociation in energy conversion.

Covalent Binding of Bone Morphogenetic Protein-2 and Transforming Growth Factor-ß3 to 3D Plotted Scaffolds for Osteochondral Tissue Regeneration.

Engineering the osteochondral tissue presents some challenges mainly relying in its function of transition from the subchondral bone to articular cartilage and the gradual variation in several biological, mechanical, and structural features. A possible solution for osteochondral regeneration might be the design and fabrication of scaffolds presenting a gradient able to mimic this transition. Covalent binding of biological factors proved to enhance cell adhesion and differentiation in two-dimensional culture substrates. Here, we used polymer brushes as selective linkers of bone morphogenetic protein-2 (BMP-2) and transforming growth factor-ß3 (TGF-ß3) on the surface of 3D scaffolds fabricated via additive manufacturing (AM) and subsequent controlled radical polymerization. These growth factors (GFs) are known to stimulate the differentiation of human mesenchymal stromal cells (hMSCs) toward the osteogenic and chondrogenic lineages, respectively. BMP-2 and TGF-ß3 were covalently bound both homogeneously within a poly(ethylene glycol) (PEG)-based brush-functionalized scaffolds, and following a gradient composition by varying their concentration along the axial section of the 3D constructs. Following an approach previously developed by our group and proved to be successful to generate fibronectin gradients, opposite brush-supported gradients of BMP-2 and TGF-ß3 were finally generated and subsequently tested to differentiate cells in a gradient fashion. The brush-supported GFs significantly influenced hMSCs osteochondral differentiation when the scaffolds were homogenously modified, yet no effect was observed in the gradient scaffolds. Therefore, this technique seems promising to maintain the biological activity of growth factors covalently linked to 3D scaffolds, but needs to be further optimized in case biological gradients are desired.