Three-Dimensional Printed Cell Culture Model Based on Spherical Colloidal Lignin Particles and Cellulose Nanofibril-Alginate Hydrogel.

Three-dimensional (3D) printing has been an emerging technique to fabricate precise scaffolds for biomedical applications. Cellulose nanofibril (CNF) hydrogels have attracted considerable attention as a material for 3D printing because of their shear-thinning properties. Combining cellulose nanofibril hydrogels with alginate is an effective method to enable cross-linking of the printed scaffolds in the presence of Ca2+ ions. In this work, spherical colloidal lignin particles (CLPs, also known as spherical lignin nanoparticles) were used to prepare CNF-alginate-CLP nanocomposite scaffolds. High-resolution images obtained by atomic force microscopy (AFM) showed that CLPs were homogeneously mixed with the CNF hydrogel. CLPs brought antioxidant properties to the CNF-alginate-CLP scaffolds in a concentration-dependent manner and increased the viscosity of the hydrogels at a low shear rate, which correspondingly provide better shape fidelity and printing resolution to the scaffolds. Interestingly, the CLPs did not affect the viscosity at high shear rates, showing that the shear thinning behavior typical for CNF hydrogels was retained, enabling easy printing. The CNF-alginate-CLP scaffolds demonstrated shape stability after printing, cross-linking, and storage in Dulbecco's phosphate buffer solution (DPBS +) containing Ca2+ and Mg2+ ions, up to 7 days. The 3D-printed scaffolds showed relative rehydration ratio values above 80% after freeze-drying, demonstrating a high water-retaining capability. Cell viability tests using hepatocellular carcinoma cell line HepG2 showed no negative effect of CLPs on cell proliferation. Fluorescence microscopy indicated that HepG2 cells grew not only on the surfaces but also inside the porous scaffolds. Overall, our results demonstrate that nanocomposite CNF-alginate-CLP scaffolds have high potential in soft-tissue engineering and regenerative-medicine applications.

Tannins of Conifer Bark as Nordic Piquancy-Sustainable Preservative and Aroma?

Bark of Norway spruce and Scots pine trees contain large amounts of condensed tannins. Tannins extracted with hot water could be used in different applications as they possess antioxidative and antimicrobial activities. The use of bark tannins as e.g., food preservatives calls for increases in our knowledge of their antioxidative activities when applied in foodstuffs. To assess the ability of bark tannins to prevent lipid oxidation, hot water extracts were evaluated in a liposome model. Isolated tannins were also applied in dry-cured, salty meat snacks either as liquid extracts or in dry-powder form. Consumer acceptance of the snacks was tested by a sensory evaluation panel where outlook, odor, taste, and structure of the snacks were evaluated and compared to a commercial product without tannin ingredients. Our results show that conifer bark tannin-rich extracts have high capacity to prevent lipid oxidation in the liposome model. The efficacies of pine and spruce bark extracts were ten to hundred folds higher, respectively, than those of phenolic berry extracts. The bark extracts did not significantly influence the odor or taste of the meat snacks. The findings indicate that bark extracts may be used as sustainable food ingredients. However, more research is needed to verify their safety.

Structural Basis of Human Parechovirus Neutralization by Human Monoclonal Antibodies.

UNLABELLED: Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has started to emerge. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases and has given moderate success. Direct inhibition of parechovirus infection using monoclonal antibodies is a potential treatment. We have developed two neutralizing monoclonal antibodies against HPeV1 and HPeV2, namely, AM18 and AM28, which also cross-neutralize other viruses. Here, we present the mapping of their epitopes using peptide scanning, surface plasmon resonance, fluorescence-based thermal shift assays, electron cryomicroscopy, and image reconstruction. We determined by peptide scanning and surface plasmon resonance that AM18 recognizes a linear epitope motif including the arginine-glycine-aspartic acid on the C terminus of capsid protein VP1. This epitope is normally used by the virus to attach to host cell surface integrins during entry and is found in 3 other viruses that AM18 neutralizes. Therefore, AM18 is likely to cause virus neutralization by aggregation and by blocking integrin binding to the capsid. Further, we show by electron cryomicroscopy, three-dimensional reconstruction, and pseudoatomic model fitting that ordered RNA interacts with HPeV1 VP1 and VP3. AM28 recognizes quaternary epitopes on the capsid composed of VP0 and VP3 loops from neighboring pentamers, thereby increasing the RNA accessibility temperature for the virus-AM28 complex compared to the virus alone. Thus, inhibition of RNA uncoating probably contributes to neutralization by AM28. IMPORTANCE: Human parechoviruses can cause mild infections to severe diseases in young children, such as neonatal sepsis, encephalitis, and cardiomyopathy. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases. In order to develop more targeted treatment, we have searched for human monoclonal antibodies that would neutralize human parechoviruses 1 and 2, associated with mild infections such as gastroenteritis and severe infections of the central nervous system, and thus allow safe treatment. In the current study, we show how two such promising antibodies interact with the virus, modeling the atomic interactions between the virus and the antibody to propose how neutralization occurs. Both antibodies can cause aggregation; in addition, one antibody interferes with the virus recognizing its target cell, while the other, recognizing only the whole virus, inhibits the genome uncoating and replication in the cell.

Effect of PEG-PDMAEMA Block Copolymer Architecture on Polyelectrolyte Complex Formation with Heparin.

Heparin is a naturally occurring polyelectrolyte consisting of a sulfated polysaccharide backbone. It is widely used as an anticoagulant during major surgical operations. However, the associated bleeding risks require rapid neutralization after the operation. The only clinically approved antidote for heparin is protamine sulfate, which is, however, ineffective against low molecular weight heparin and can cause severe adverse reactions in patients. In this study, the facile synthesis of cationic-neutral diblock copolymers and their effective heparin binding is presented. Poly(ethylene glycol)-poly(2-(dimethylamino)ethyl methacrylate) (PEG-PDMAEMA) block copolymers were synthesized in two steps via atom-transfer radical polymerization (ATRP) using PEG as a macroinitiator. Solution state binding between heparin and a range of PEG-PDMAEMA block copolymers and one homopolymer was studied with dynamic light scattering and methylene blue displacement assay. Also in vitro binding in plasma was studied by utilizing a chromogenic heparin anti-Xa assay. Additionally, quartz crystal microbalance and multiparametric surface plasmon resonance were used to study the surface adsorption kinetics of the polymers on a heparin layer. It was shown that the block copolymers and heparin form electrostatically bound complexes with varying colloidal properties, where the block lengths play a key role in controlling the heparin binding affinity, polyelectrolyte complex size and surface charge. With the optimized polymers (PEG114PDMAEMA52 and PEG114PDMAEMA100), heparin could be neutralized in a dose-dependent manner, and bound efficiently into small neutral complexes, with a hydrodynamic radius less than 100 nm. These complexes had only a limited effect on cell viability. Based on these studies, our approach paves the way for the development of new polymeric heparin binding agents.

Hierarchically ordered supramolecular protein-polymer composites with thermoresponsive properties.

Synthetic macromolecules that can bind and co-assemble with proteins are important for the future development of biohybrid materials. Active systems are further required to create materials that can respond and change their behavior in response to external stimuli. Here we report that stimuli-responsive linear-branched diblock copolymers consisting of a cationic multivalent dendron with a linear thermoresponsive polymer tail at the focal point, can bind and complex Pyrococcus furiosus ferritin protein cages into crystalline arrays. The multivalent dendron structure utilizes cationic spermine units to bind electrostatically on the surface of the negatively charged ferritin cage and the in situ polymerized poly(di(ethylene glycol) methyl ether methacrylate) linear block enables control with temperature. Cloud point of the final product was determined with dynamic light scattering (DLS), and it was shown to be approximately 31 °C at a concentration of 150 mg/L. Complexation of the polymer binder and apoferritin was studied with DLS, small-angle X-ray scattering, and transmission electron microscopy, which showed the presence of crystalline arrays of ferritin cages with a face-centered cubic (fcc, Fm3m)) Bravais lattice where lattice parameter a=18.6 nm. The complexation process was not temperature dependent but the final complexes had thermoresponsive characteristics with negative thermal expansion.

Electron cryotomography of measles virus reveals how matrix protein coats the ribonucleocapsid within intact virions.

Measles virus is a highly infectious, enveloped, pleomorphic virus. We combined electron cryotomography with subvolume averaging and immunosorbent electron microscopy to characterize the 3D ultrastructure of the virion. We show that the matrix protein forms helices coating the helical ribonucleocapsid rather than coating the inner leaflet of the membrane, as previously thought. The ribonucleocapsid is folded into tight bundles through matrix-matrix interactions. The implications for virus assembly are that the matrix already tightly interacts with the ribonucleocapsid in the cytoplasm, providing a structural basis for the previously observed regulation of RNA transcription by the matrix protein. Next, the matrix-covered ribonucleocapsids are transported to the plasma membrane, where the matrix interacts with the envelope glycoproteins during budding. These results are relevant to the nucleocapsid organization and budding of other paramyxoviruses, where isolated matrix has been observed to form helices.

Inspired by nature: Fiber networks functionalized with tannic acid and condensed tannin-rich extracts of Norway spruce bark show antimicrobial efficacy.

This study demonstrated the antibacterial and antiviral potential of condensed tannins and tannic acid when incorporated into fiber networks tested for functional material purposes. Condensed tannins were extracted from industrial bark of Norway spruce by using pressurized hot water extraction (PHWE), followed by purification of extracts by using XADHP7 treatment to obtain sugar-free extract. The chemical composition of the extracts was analyzed by using HPLC, GC‒MS and UHPLC after thiolytic degradation. The test matrices, i.e., lignocellulosic handsheets, were produced and impregnated with tannin-rich extracts, and tannic acid was used as a commercial reference. The antibacterial and antiviral efficacy of the handsheets were analyzed by using bioluminescent bacterial strains (Staphylococcus aureus RN4220+pAT19 and Escherichia coli K12+pCGLS11) and Enterovirus coxsackievirus B3. Potential bonding of the tannin-rich extract and tannic acid within the fiber matrices was studied by using FTIR-ATR spectroscopy. The deposition characteristics (distribution and accumulation patterns) of tannin compounds and extracts within fiber networks were measured and visualized by direct chemical mapping using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and digital microscopy. Our results demonstrated for the first time, how tannin-rich extracts obtained from spruce bark side streams with green chemistry possess antiviral and antibacterial properties when immobilized into fiber matrices to create substitutes for plastic hygienic products, personal protection materials such as surgical face masks, or food packaging materials to prolong the shelf life of foodstuffs and prevent the spread of infections. However, more research is needed to further develop this proof-of-concept to ensure stable chemical bonding in product prototypes with specific chemistry.

Efficient IgM assembly and secretion require the plasma cell induced endoplasmic reticulum protein pERp1.

Plasma cells daily secrete their own mass in antibodies, which fold and assemble in the endoplasmic reticulum (ER). To reach these levels, cells require pERp1, a novel lymphocyte-specific small ER-resident protein, which attains expression levels as high as BiP when B cells differentiate into plasma cells. Although pERp1 has no homology with known ER proteins, it does contain a CXXC motif typical for oxidoreductases. In steady state, the CXXC cysteines are locked by two parallel disulfide bonds with a downstream C(X)(6)C motif, and pERp1 displays only modest oxidoreductase activity. pERp1 emerged as a dedicated folding factor for IgM, associating with both heavy and light chains and promoting assembly and secretion of mature IgM.

Crystallization and preliminary crystallographic analysis of mouse peroxiredoxin II with significant pseudosymmetry.

Peroxiredoxin II was cloned from mouse B cells into pCold 1 expression vector and produced as a His-tagged recombinant protein in Escherichia coli. A ring form was isolated by gel filtration. A crystal obtained by the sitting-drop vapour-diffusion method diffracted to 1.77 A resolution at 100 K. The crystal belonged to space group P2(1)2(1)2, with unit-cell parameters a = 117.4, b = 133.9, c = 139.1 A. The asymmetric unit is expected to contain six dimers of peroxiredoxin II, with a corresponding solvent content of 39.3%. Peaks in the native Patterson function together with pseudo-systematic absences suggested that the crystals suffered from severe translational pseudosymmetry.

CD2-associated protein is widely expressed and differentially regulated during embryonic development.

CD2-associated protein (CD2AP) is an adapter protein that is involved in various signaling and vesicular trafficking processes and also functions as a linker between plasma membrane proteins and the actin cytoskeleton. The protein is known to have important functions in T cells and glomerular podocytes, but it is also expressed by many other adult-type tissues and cells. Here we analyzed the expression of the protein during early embryonic development and organogenesis of the mouse. The results showed differential tissue-specific regulation of CD2AP in developing and maturing organs. In oocytes and pre-implantation embryos, CD2AP was located diffusely in the cytoplasm, whereas in late blastocysts it was concentrated to the intercellular contacts. During organogenesis, CD2AP was distinctly upregulated upon, e.g., the pretubular aggregation of metanephric mesenchyme cells and the appearance of the osteoblastic rim around cartilages during endochondral ossification. High CD2AP expression was also observed during epithelial-like conversion of some highly specialized secretory cell types such as the odontoblasts, the cells of the choroid plexus and the decidualized cells of the endometrial stroma. In other instances, such as the development of the proximal tubuli of the kidney and the flat alveolar epithelium of the lung, the protein was downregulated upon differentiation and maturation of the cells. Finally, certain cells, e.g., glomerular podocytes, those forming the collecting ducts of the kidney, and the urothelium of the kidney pelvis, expressed CD2AP throughout their differentiation and maturation. Multiple molecules and complex pathways regulate embryogenesis, and scaffolding proteins apparently have pivotal roles in targeting and finetuning, e.g., growth factor- or hormone-induced processes. The cell-type specific spatio-temporal regulation of CD2AP during development suggests that this adapter protein is a key regulatory partner in many signaling pathways and cellular processes governing differentiation and morphogenesis.

Cooperative colloidal self-assembly of metal-protein superlattice wires.

Material properties depend critically on the packing and order of constituent units throughout length scales. Beyond classically explored molecular self-assembly, structure formation in the nanoparticle and colloidal length scales have recently been actively explored for new functions. Structure of colloidal assemblies depends strongly on the assembly process, and higher structural control can be reliably achieved only if the process is deterministic. Here we show that self-assembly of cationic spherical metal nanoparticles and anionic rod-like viruses yields well-defined binary superlattice wires. The superlattice structures are explained by a cooperative assembly pathway that proceeds in a zipper-like manner after nucleation. Curiously, the formed superstructure shows right-handed helical twisting due to the right-handed structure of the virus. This leads to structure-dependent chiral plasmonic function of the material. The work highlights the importance of well-defined colloidal units when pursuing unforeseen and complex assemblies.Colloidal self-assembly is a unique method to produce three-dimensional materials with well-defined hierarchical structures and functionalities. Liljeström et al. show controlled preparation of macroscopic chiral wires with helical plasmonic superlattice structure composed of metal nanoparticles and viruses.

Erratum: Genomic RNA folding mediates assembly of human parechovirus.

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Genomic RNA folding mediates assembly of human parechovirus.

Assembly of the major viral pathogens of the Picornaviridae family is poorly understood. Human parechovirus 1 is an example of such viruses that contains 60 short regions of ordered RNA density making identical contacts with the protein shell. We show here via a combination of RNA-based systematic evolution of ligands by exponential enrichment, bioinformatics analysis and reverse genetics that these RNA segments are bound to the coat proteins in a sequence-specific manner. Disruption of either the RNA coat protein recognition motif or its contact amino acid residues is deleterious for viral assembly. The data are consistent with RNA packaging signals playing essential roles in virion assembly. Their binding sites on the coat proteins are evolutionarily conserved across the Parechovirus genus, suggesting that they represent potential broad-spectrum anti-viral targets.The mechanism underlying packaging of genomic RNA into viral particles is not well understood for human parechoviruses. Here the authors identify short RNA motifs in the parechovirus genome that bind capsid proteins, providing approximately 60 specific interactions for virion assembly.

Cellular delivery of enzyme-loaded DNA origami.

In this communication, we show that active enzymes can be delivered into HEK293 cells in vitro when they are attached to tubular DNA origami nanostructures. We use bioluminescent enzymes as a cargo and monitor their activity from a cell lysate. The results show that the enzymes stay intact and retain their activity in the transfection process. The method is highly modular, which makes it a compelling candidate for a great variety of delivery applications.

Cationic polymers for DNA origami coating - examining their binding efficiency and tuning the enzymatic reaction rates.

DNA origamis are fully tailored, programmable, biocompatible and readily functionalizable nanostructures that provide an excellent foundation for the development of sophisticated drug-delivery systems. However, the DNA origami objects suffer from certain drawbacks such as low cell-transfection rates and low stability. A great deal of studies on polymer-based transfection agents, mainly focusing on polyplex formation and toxicity, exists. In this study, the electrostatic binding between a brick-like DNA origami and cationic block-copolymers was explored. The effect of the polymer structure on the binding was investigated and the toxicity of the polymer-origami complexes evaluated. The study shows that all of the analyzed polymers had a suitable binding efficiency irrespective of the block structure. It was also observed that the toxicity of polymer-origami complexes was insignificant at the biologically relevant concentration levels. Besides brick-like DNA origamis, tubular origami carriers equipped with enzymes were also coated with the polymers. By adjusting the amount of cationic polymers that cover the DNA structures, we showed that it is possible to control the enzyme kinetics of the complexes. This work gives a starting point for further development of biocompatible and effective polycation-based block copolymers that can be used in coating different DNA origami nanostructures for various bioapplications.

DNA Nanostructures as Smart Drug-Delivery Vehicles and Molecular Devices.

DNA molecules can be assembled into custom predesigned shapes via hybridization of sequence-complementary domains. The folded structures have high spatial addressability and a tremendous potential to serve as platforms and active components in a plethora of bionanotechnological applications. DNA is a truly programmable material, and its nanoscale engineering thus opens up numerous attractive possibilities to develop novel methods for therapeutics. The tailored molecular devices could be used in targeting cells and triggering the cellular actions in the biological environment. In this review we focus on the DNA-based assemblies - primarily DNA origami nanostructures - that could perform complex tasks in cells and serve as smart drug-delivery vehicles in, for example, cancer therapy, prodrug medication, and enzyme replacement therapy.

Association between the intrinsically disordered protein PEX19 and PEX3.

In peroxisomes, peroxins (PEXs) 3 and 19 are the principal protein components of the machinery required for early peroxisomal biogenesis. For further insight into the interaction of PEX3 and PEX19, we used hydrogen exchange mass spectrometry to monitor conformational changes during complex formation between PEX3 and PEX19 in vitro. Our data showed that PEX19 remained highly flexible during interaction with PEX3. However, we could detect three changes, one each in the N-and C-terminus along with a small stretch in the middle of PEX19 (F64-L74) which became shielded from hydrogen exchange when interacting with PEX3. PEX3 became more protected from hydrogen exchange in the binding groove for PEX19 with only small changes elsewhere. Most likely the N-terminus of PEX19 initiates the binding to PEX3, and then subtle conformational changes in PEX3 affect the surface of the PEX3 molecule. PEX19 in turn, is stabilized by folding of a short helix and its C-terminal folding core permitting PEX19 to bind to PEX3 with higher affinity than just the N-terminal interaction allows. Thus within the cell, PEX3 is stabilized by PEX19 preventing PEX3 aggregation.

The high resolution NMR structure of the third SH3 domain of CD2AP.

CD2 associated protein (CD2AP) is an adaptor protein that plays an important role in cell to cell union needed for the kidney function. CD2AP interacts, as an adaptor protein, with different natural targets, such as CD2, nefrin, c-Cbl and podocin. These proteins are believed to interact to one of the three SH3 domains that are positioned in the N-terminal region of CD2AP. To understand the network of interactions between the natural targets and the three SH3 domains (SH3-A, B and C), we have started to determine the structures of the individual SH3 domains. Here we present the high-resolution structure of the SH3-C domain derived from NMR data. Full backbone and side-chain assignments were obtained from triple-resonance spectra. The structure was determined from distance restraints derived from high-resolution 600 and 800 MHz NOESY spectra, together with phi and psi torsion angle restraints based on the analysis of 1HN, 15N, 1Halpha, 13Calpha, 13CO and 13Cbeta chemical shifts. Structures were calculated using CYANA and refined in water using RECOORD. The three-dimensional structure of CD2AP SH3-C contains all the features that are typically found in other SH3 domains, including the general binding site for the recognition of polyproline sequences.

Interaction of endogenous nephrin and CD2-associated protein in mouse epithelial M-1 cell line.

The interpodocyte slit diaphragm is an essential structure for maintaining the functional glomerular filtration barrier. The slit diaphragm is proposed to consist of an interacting meshwork of nephrin molecules. Earlier studies with tagged proteins have suggested that the intracellular part of nephrin interacts with CD2-associated protein (CD2AP). This study was addressed to show by coimmunoprecipitation and pulldown assays an interaction of endogenously expressed nephrin and CD2AP in the kidney-derived mouse epithelial M-1 cell line, to provide evidence of the domain(s) of CD2AP involved in the interaction, and to show the localization of the respective proteins by immunoelectron microscopy in kidney cortex. In addition, the localization of CD2AP, podocin, alpha-actinin 4, and nephrin was studied in human kidney glomeruli and in M-1 cells by immunofluorescence microscopy. The results indicate an endogenous interaction between nephrin and CD2AP in M-1 cells and suggest that this interaction is mediated by the third Src homology 3 (SH3) domain of CD2AP. We also show by immunoelectron microscopy that nephrin and CD2AP are detected at the slit diaphragm area, supporting their interaction in the glomeruli in vivo. In addition, nephrin was found to partially colocalize with CD2AP and podocin in double immunofluorescence microscopy, confirming the close proximity of these proteins and proposing that these proteins may belong to nephrin-associated protein complex in glomeruli. The existence of nephrin, CD2AP, podocin, and alpha-actinin 4 enables further characterization of their relationship in M-1 cells.