Association of 2-oxoacid dehydrogenase complexes with respirasomes in mitochondria.

In the present study, cryo-electron tomography was used to investigate the localization of 2-oxoacid dehydrogenase complexes (OADCs) in cardiac mitochondria and mitochondrial inner membrane samples. Two classes of ordered OADC inner cores with different symmetries were distinguished and their quaternary structures modeled. One class corresponds to pyruvate dehydrogenase complexes and the other to dehydrogenase complexes of α-ketoglutarate and branched-chain α-ketoacids. OADCs were shown to be localized in close proximity to membrane-embedded respirasomes, as observed both in densely packed lamellar cristae of cardiac mitochondria and in ruptured mitochondrial samples where the dense packing is absent. This suggests the specificity of the OADC-respirasome interaction, which allows localized NADH/NAD+ exchange between OADCs and complex I of the respiratory chain. The importance of this local coupling is based on OADCs being the link between respiration, glycolysis and amino acid metabolism. The coupling of these basic metabolic processes can vary in different tissues and conditions and may be involved in the development of various pathologies. The present study shows that this important and previously missing parameter of mitochondrial complex coupling can be successfully assessed using cryo-electron tomography.

Cryo-electron microscopy of adipose tissue extracellular vesicles in obesity and type 2 diabetes mellitus.

Extracellular vesicles (EVs) are cell-derived membrane vesicles which play an important role in cell-to-cell communication and physiology. EVs deliver biological information from producing to recipient cells by transport of different cargo such as proteins, mRNAs, microRNAs, non-coding RNAs and lipids. Adipose tissue EVs could regulate metabolic and inflammatory interactions inside adipose tissue depots as well as distal tissues. Thus, adipose tissue EVs are assumed to be implicated in obesity-associated pathologies, notably in insulin resistance and type 2 diabetes mellitus (T2DM). In this study we for the first time characterize EVs secreted by visceral (VAT) and subcutaneous adipose tissue (SAT) of patients with obesity and T2DM with standard methods as well as analyze their morphology with cryo-electron microscopy. Cryo-electron microscopy allowed us to visualize heterogeneous population of EVs of various size and morphology including single EVs and EVs with internal membrane structures in samples from obese patients as well from the control group. Single vesicles prevailed (up to 85% for SAT, up to 75% for VAT) and higher proportion of EVs with internal membrane structures compared to SAT was typical for VAT. Decreased size of single and double SAT EVs compared to VAT EVs, large proportion of multilayered EVs and all EVs with internal membrane structures secreted by VAT distinguished obese patients with/without T2DM from the control group. These findings could support the idea of modified biogenesis of EVs during obesity and T2DM.

Membrane-mediated interaction of non-conventional snake three-finger toxins with nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptor of α7 type (α7-nAChR) presented in the nervous and immune systems and epithelium is a promising therapeutic target for cognitive disfunctions and cancer treatment. Weak toxin from Naja kaouthia venom (WTX) is a non-conventional three-finger neurotoxin, targeting α7-nAChR with weak affinity. There are no data on interaction mode of non-conventional neurotoxins with nAChRs. Using α-bungarotoxin (classical three-finger neurotoxin with high affinity to α7-nAChR), we showed applicability of cryo-EM to study complexes of α7-nAChR extracellular ligand-binding domain (α7-ECD) with toxins. Using cryo-EM structure of the α7-ECD/WTX complex, together with NMR data on membrane active site in the WTX molecule and mutagenesis data, we reconstruct the structure of α7-nAChR/WTX complex in the membrane environment. WTX interacts at the entrance to the orthosteric site located at the receptor intersubunit interface and simultaneously forms the contacts with the membrane surface. WTX interaction mode with α7-nAChR significantly differs from α-bungarotoxin's one, which does not contact the membrane. Our study reveals the important role of the membrane for interaction of non-conventional neurotoxins with the nicotinic receptors.

The Performance of Nonwoven PLLA Scaffolds of Different Thickness for Stem Cells Seeding and Implantation.

The 3D reconstruction of 100 µm- and 600 µm-thick fibrous poly-L/L-lactide scaffolds was performed by confocal laser scanning microscopy and supported by scanning electron microscopy and showed that the density of the fibers on the side adjacent to the electrode is higher, which can affect cell diffusion, while the pore size is generally the same. Bone marrow mesenchymal stem cells cultured in a 600 µm-thick scaffold formed colonies and produced conditions for cell differentiation. An in vitro study of stem cells after 7 days revealed that cell proliferation and hepatocyte growth factor release in the 600 µm-thick scaffold were higher than in the 100 µm-thick scaffold. An in vivo study of scaffolds with and without stem cells implanted subcutaneously onto the backs of recipient mice was carried out to test their biodegradation and biocompatibility over a 0-3-week period. The cells seeded onto the 600 µm-thick scaffold promoted significant neovascularization in vivo. After 3 weeks, a significant number of donor cells persisted only on the inside of the 600 µm-thick scaffold. Thus, the use of bulkier matrices allows to prolong the effect of secretion of growth factors by stem cells during implantation. These 600 µm-thick scaffolds could potentially be utilized to repair and regenerate injuries with stem cell co-culture for vascularization of implant.

Nanomechanical characterization of exosomes and concomitant nanoparticles from blood plasma by PeakForce AFM in liquid.

BACKGROUND: To date, EVs characterization techniques are extremely diverse. The contribution of AFM, in particular, is often confined to size distribution. While AFM provides a unique possibility to carry out measurements in situ, nanomechanical characterization of EVs is still missing. METHODS: Blood plasma EVs were isolated by ultracentrifugation, analyzed by flow cytometry and NTA. Followed by cryo-EM, we applied PeakForce AFM to assess morphological and nanomechanical properties of EVs in liquid. RESULTS: Nanoparticles were subdivided by their size estimated for their suspended state into sub-sets of small S1-EVs (< 30 nm), S2-EVs (30-50 nm), and sub-set of large ones L-EVs (50-170 nm). Non-membranous S1-EVs were distinguished by higher Young's modulus (10.33(7.36;15.25) MPa) and were less deformed by AFM tip (3.6(2.8;4.4) nm) compared to membrane exosomes S2-EVs (6.25(4.52;8.24) MPa and 4.8(4.3;5.9) nm). L-EVs were identified as large membrane exosomes, heterogeneous by their nanomechanical properties (22.43(8.26;53.11) MPa and 3.57(2.07;7.89) nm). Nanomechanical mapping revealed a few non-deformed L-EVs, of which Young's modulus rose up to 300 MPa. Taken together with cryo-EM, these results lead us to the suggestion that two or more vesicles could be contained inside a large one being a multilayer vesicle. CONCLUSIONS: We identified particles similar in morphology and showed differences in nanomechanical properties that could be attributed to the features of their inner structure. GENERAL SIGNIFICANCE: Our results further elucidate the identification of EVs and concomitant nanoparticles based on their nanomechanical properties.

Development of Submicrocapsules Based on Co-Assembled Like-Charged Silica Nanoparticles and Detonation Nanodiamonds and Polyelectrolyte Layers.

Capsules with shells based on nanoparticles of different nature co-assembled at the interface of liquid phases of emulsion are promising carriers of lipophilic drugs. To obtain such capsules, theoretically using the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory and experimentally using dynamic light-scattering (DLS) and transmission electron microscopy (TEM) methods, the interaction of like-charged silica nanoparticles and detonation nanodiamonds in an aqueous solution was studied and their ratios selected for the formation of submicron-sized colloidosomes. The resulting colloidosomes were modified with additional layers of nanoparticles and polyelectrolytes, applying LbL technology. As a model anti-cancer drug, thymoquinone was loaded into the developed capsules, demonstrating a significant delay of the release as a result of colloidosome surface modification. Fluorescence flow cytometry and confocal laser scanning microscopy showed efficient internalization of the capsules by MCF7 cancer cells. The obtained results demonstrated a high potential for nanomedicine application in the field of the drug-delivery system development.

Vaccine building 'kit': combining peptide bricks to elicit a desired immune response without adding an adjuvant.

Protein nanoparticles (NPs) can be used as vaccine platforms for target antigen presentation. Aim: To conduct a proof-of-concept study to demonstrate that an effective NP platform can be built based on a short self-assembling peptide (SAP) rather than a large self-assembling protein. Materials & methods: SUMO-based protein fusions (SFs) containing an N-terminal SAP and a C-terminal antigen were designed, expressed in Escherichia coli and purified. The structure was investigated by electron microscopy. The antibody response was tested in mice after two adjuvant-free immunizations. Results: Renatured SFs form fiber-like NPs with the antigen exposed on the surface and induce a significant antibody response with a remarkably high target-to-platform ratio. Conclusion: The platform is effective and has considerable potential for modification toward various applications, including vaccine development.

We aimed to extend the arsenal of protein platforms used for vaccine development. To this end, in this proof-of-concept study we constructed new self-assembling fusion proteins consisting of three modules. Module 1 is responsible for the self-assembly, while modules 2 and 3 are responsible for the immune response. Modules 1 and 2 form the platform, while module 3 represents the target antigen exposed on the surface of the self-assembled nanoparticles. After conventional biosynthesis in Escherichia coli, the proteins undergo efficient self-assembly during purification, and the resulting nanoparticles elicit a strong immune response without using an enhancing agent (adjuvant). The simple modular design and a high target-to-platform ratio of the immune response make our system a promising approach for practical applications, including vaccine development.

Printable Alginate Hydrogels with Embedded Network of Halloysite Nanotubes: Effect of Polymer Cross-Linking on Rheological Properties and Microstructure.

Rapidly growing 3D printing of hydrogels requires network materials which combine enhanced mechanical properties and printability. One of the most promising approaches to strengthen the hydrogels consists of the incorporation of inorganic fillers. In this paper, the rheological properties important for 3D printability were studied for nanocomposite hydrogels based on a rigid network of percolating halloysite nanotubes embedded in a soft alginate network cross-linked by calcium ions. Particular attention was paid to the effect of polymer cross-linking on these properties. It was revealed that the system possessed a pronounced shear-thinning behavior accompanied by a viscosity drop of 4-5 orders of magnitude. The polymer cross-links enhanced the shear-thinning properties and accelerated the viscosity recovery at rest so that the system could regain 96% of viscosity in only 18 s. Increasing the cross-linking of the soft network also enhanced the storage modulus of the nanocomposite system by up to 2 kPa. Through SAXS data, it was shown that at cross-linking, the junction zones consisting of fragments of two laterally aligned polymer chains were formed, which should have provided additional strength to the hydrogel. At the same time, the cross-linking of the soft network only slightly affected the yield stress, which seemed to be mainly determined by the rigid percolation network of nanotubes and reached 327 Pa. These properties make the alginate/halloysite hydrogels very promising for 3D printing, in particular, for biomedical purposes taking into account the natural origin, low toxicity, and good biocompatibility of both components.

Magnetic-field-assisted synthesis of anisotropic iron oxide particles: Effect of pH.

The synthesis of magnetite (Fe3O4) nanorods using reverse co-precipitation of Fe3+ and Fe2+ ions in the presence of a static magnetic field is reported in this work. The phase composition and crystal structure of the synthesized material were investigated using electron diffraction, Raman spectroscopy, and transmission electron microscopy. It was shown that the morphology of the reaction product strongly depends on the amount of OH- ions in the reaction mixture, varying from Fe3O4 nanorods to spherical Fe3O4 nanoparticles. Fe3O4 nanorods were examined using high-resolution transmission electron microscopy proving that they are single-crystalline and do not have any preferred crystallographic orientation along the axis of the rods. According to the data obtained a growth mechanism was proposed for the rods that consists of the dipole-dipole interaction between their building blocks (small hexagonal faceted magnetite nanocrystals), which are formed during the first step of the reaction. The study suggests a facile, green and controllable method for synthesizing anisotropic magnetic nanoparticles in the absence of stabilizers, which is important for further modification of their surfaces and/or incorporation of the nanoparticles into different media.

Biodegradable poly(l-lactide)/calcium phosphate composites with improved properties for orthopedics: Effect of filler and polymer crystallinity.

Biodegradable poly(l-lactide)/calcium phosphate composites are promising materials for fabrication of bone fixation implants with improved properties. Multistage compounding was proposed as an efficient method for the preparation of biodegradable poly(l-lactide)/calcium phosphate composites with submicron filler dispersion and mechanical characteristics similar to native bone. The improvement of the characteristics is caused both by the filler itself and by the increase of polymer crystallinity due to the nucleation effect. The technique allows to fabricate biodegradable composites with controlled properties by varying concentration and type of the filler as well as degree of PLLA matrix crystallinity. Animal studies revealed that all the composites were biocompatible and non-toxic.

Electroconductive PEDOT:PSS-based hydrogel prepared by freezing-thawing method.

Biopolymer-based composition with adding of conductive polymer poly-(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT PSS) was made by mixing of iota-carrageenan (CRG), polyvinyl alcohol (PVA) and PEDOT PSS followed by freezing/thawing cycles. The method is environmentally friendly and based on the formation of polymer matrix upon of mixing CRG, PVA and PEDOT PSS and formation of porous physical gel due to freezing/thawing cycles. It is necessary to mention that all components are well-known as biocompatible materials. The resulting material is stable in water and also has swelling capability both in distilled water and physiological solutions. Structure of material was characterized by means of X-ray diffraction, optical and electron microscopy. Electrophysical investigations also were performed. The conductivity of the gel immersed in distilled water is comparable with the dry gel value and close to 0.01 [S/cm].

Protective Dps-DNA co-crystallization in stressed cells: an in vitro structural study by small-angle X-ray scattering and cryo-electron tomography.

Under severe or prolonged stress, bacteria produce a nonspecific DNA-binding protein (Dps), which effectively protects DNA against damaging agents both in vitro and in vivo by forming intracellular biocrystals. The phenomenon of protective crystallization of DNA in living cells has been intensively investigated during the last two decades; however, the results of studies are somewhat contradictory, and up to now, there has been no direct determination of a Dps-DNA crystal structure. Here, we report the in vitro analysis of the vital process of Dps-DNA co-crystallization using two complementary structural methods: synchrotron small-angle X-ray scattering in solution and cryo-electron tomography. Importantly, for the first time, the DNA in the co-crystals was visualized, and the lattice parameters of the crystalline Dps-DNA complex were determined.

Non-woven bilayered biodegradable chitosan-gelatin-polylactide scaffold for bioengineering of tracheal epithelium.

OBJECTIVES: The conversion of tissue engineering into a routine clinical tool cannot be achieved without a deep understanding of the interaction between cells and scaffolds during the process of tissue formation in an artificial environment. Here, we have investigated the cultivation conditions and structural features of the biodegradable non-woven material in order to obtain a well-differentiated human airway epithelium. MATERIALS AND METHODS: The bilayered scaffold was fabricated by electrospinning technology. The efficiency of the scaffold has been evaluated using MTT cell proliferation assay, histology, immunofluorescence and electron microscopy. RESULTS: With the use of a copolymer of chitosan-gelatin-poly-l-lactide, a bilayered non-woven scaffold was generated and characterized. The optimal structural parameters of both layers for cell proliferation and differentiation were determined. The basal airway epithelial cells differentiated into ciliary and goblet cells and formed pseudostratified epithelial layer on the surface of the scaffold. In addition, keratinocytes formed a skin equivalent when seeded on the same scaffold. A comparative analysis of growth and differentiation for both types of epithelium was performed. CONCLUSIONS: The structural parameters of nanofibres should be selected experimentally depending on polymer composition. The major challenges on the way to obtain the well-differentiated equivalent of respiratory epithelium on non-woven scaffold include the following: the balance between scaffold permeability and thickness, proper combination of synthetic and natural components, and culture conditions sufficient for co-culturing of airway epithelial cells and fibroblasts. For generation of skin equivalent, the lack of diffusion is not so critical as for pseudostratified airway epithelium.

Control on rheological behavior of collagen 1 dispersions for efficient electrospinning.

For efficient manufacturing of fibrous collagen-based materials by electrospinning, the search on optimal rheological parameters is of the great importance. Rheological characteristics and denaturation of collagen in aqueous dispersions were studied as a function of shear rate and acetic acid concentration in the range of 3-9% w/w at temperature from 20 to 40°C. It was shown that an increase in temperature, acetic acid concentration of the collagen dispersion leads to a significant decrease in its viscosity. It was found that helical conformation of the collagen macromolecules is preserved up to 31°C. An increase in acetic acid concentration leads to a reduction of denaturation temperature. The complex viscosity of collagen dispersions exhibits a sharp drop, followed by a rapid growth of damping factor in the temperature range from 22 to 35°C. Both storage (G') and loss (G″) moduli increase with frequency and collagen concentration. It was revealed that optimal parameters for electrospinning of highly concentrated collagen dispersions can be achieved by adjusting of the concentration of acetic acid, temperature, and stirring speed. As a result, collagen nonwoven materials with diameter from 100 to 700 nm were obtained. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 312-318, 2019.

Effect of Composition and Molecular Structure of Poly(l-lactic acid)/Poly(ethylene oxide) Block Copolymers on Micellar Morphology in Aqueous Solution.

The effect of the hydrophobic block length in diblock (PLLA x- b-PEO113, x = 64, 166, 418) and triblock (PLLA y- b-PEO91- b-PLLA y, y = 30, 52, 120) copolymers of l-lactic acid and ethylene oxide on the structure of micelles prepared by dialysis was studied by wide- and small-angle X-ray scattering in dilute aqueous solution, dynamic light scattering, transmission electron microscopy, atomic force microscopy, and force spectroscopy. It was found that the size of the crystalline PLLA core is weakly dependent on the PLLA block length. In addition to individual micelles, a number of their micellar clusters were detected with characteristic distance between adjacent micelle cores decreasing with an increase in PLLA block length. This effect was explained by the change in the conformation of PEO chains forming the micellar corona because of their overcrowding. Force spectroscopy experiments also reveal a more stretched conformation of the PEO chains for the block copolymers with a shorter PLLA block. A model describing the structure of the individual micelles and their clusters was proposed.

Multi-hierarchical tissue-engineering ECM-like scaffolds based on cellulose acetate with collagen and chitosan fillers.

A novel high-tech composite biomimetic matrixes for a wide range of medical purposes were prepared. The structure of scaffolds was inspired by the architecture of native decellularized tissue: material consists of a sponge and fibrous components of different spatial geometry based on cellulose acetate with collagen or chitosan filler. The fibrous component was prepared by electrospinning, the sponge - freeze-drying technique. The influence of main technological parameters, such as freeze mode, polymer type and concentration, etc. on the fiber-sponge architecture and properties was examined. It was shown that scaffolds with different types of microstructure can be obtained employing this technique. The impregnation of chitosan or collagen filler in fiber matrix also significantly improves mechanical properties up to 40 MPa for strength and 600 MPa for Young's modulus.