DNA Condensation by Peptide-Conjugated PAMAM Dendrimers. Influence of Peptide Charge.

Nucleic acid delivery to cells is an important therapeutic strategy that requires the transport of nucleic acids to intracellular compartments and their protection from enzymatic degradation. This can be achieved through the complexation of the nucleic acids with polycations. Poly(amidoamine) (PAMAM) dendrimers and peptide-conjugated dendrimers have been investigated as delivery vectors. Inspired by these studies and the role of flexible peptide domains in protein-DNA interactions, we studied the impact of conjugating two peptides (tails) to generation 2 (G2) PAMAM dendrimers on DNA condensation and polyplex formation. Using gel electrophoresis, dye exclusion assays, atomic force microscopy, and Monte Carlo simulations, it is shown that the steric impact of neutral peptide tails is to hinder the formation of DNA-G2 polyplexes composed of multiple DNA chains. If the tails are negatively charged, which results in overall neutral G2 conjugates, then the interaction of G2 with DNA is hindered. Increasing the net positive charge of the tails resulted in the complexation capacity of G2 with the DNA being restored. While DNA complexation is obtained for a similar net charge balance for G2 and G2 conjugates with positive tails, fewer of the latter are required to achieve a comparable condensation degree. Furthermore, it is shown that about 40% of the DNA remains accessible to binding by small molecules. Overall, this shows that tuning the net charge of peptide tails conjugated to PAMAM dendrimers offers a handle to control the complexation capacity of DNA, which can be explored as a novel route for optimization as gene delivery vehicles.

ß-(1 → 3)(1 → 6)glucan from Schizophyllum commune 227E.32: High yield production via glucose/xylose co-metabolization.

A co-metabolization of xylose and glucose by Schizophyllum commune 227E.32 wild mushroom for exopolysaccharide (EPS) production is presented. Cultivations performed with S. commune 227E.32 at different xylose concentrations demonstrated that the concentration of 50 g·L-1 of xylose achieved the highest EPS production, around 4.46 g·L-1. Scale-up in a stirred tank reactor (STR) was performed. 10 % inoculum showed the highest cost/benefit ratio regarding sugar conversion and EPS production (Y P/S = 0.90 g·g-1), achieving 1.82 g·L-1 of EPS. Isolation, purification, and characterization were conducted with EPS produced in flasks and STR. GC-MS analysis showed glucose as main monosaccharide constituents for both isolates. 13C NMR and HSQC-edited showed that both EPS isolated consisted of a ß-D-Glcp (1 â†’ 3) main chain, partially substituted at O-6 with nonreducing ß-D-Glcp ends on every third residue, similar to ß-D-glucan isolated from S. commune basidiomes known as schizophyllan (SPG). The Mw was determined by GPC to 1.5 × 106 Da (flasks) and 1.1 × 106 Da (STR). AFM topographs revealed a semi-flexible appearance of the ß-D-glucan, consistent with the triple helical structures adopted by SPG and overall contour length consistent with a high molar mass.

Microfluidic dual picoinjection based encapsulation of hemoglobin in alginate microcapsules reinforced by a poly(L-lysine)-g-poly(ethylene glycol).

Hemoglobin (Hb) encapsulation inside polysaccharide hydrogels has been considered a possible red blood cell (RBC) surrogate in transfusiology. Here we report on the microfluidic dual picoinjection assisted synthesis of Hb encapsulated alginate-poly(L-lysine)-g-poly(ethylene glycol) beads. This process is realized by the on-chip injections of blended Hb alginate solutions in emulsified aqueous calcium chloride (CaCl2) droplets followed by a subsequent injection of an aqueous PLL-g-PEG into each emulsified aqueous droplet. The proposed fabrication approach was realized using a flow-focusing and two picoinjection sites in a single PDMS device. Aqueous CaCl2 solution was emulsified and infused with Hb-alginate solution as the squeezed droplet passed through the first picoinjection site. The injection of PLL-g-PEG to reinforce the microgel and minimize the protein leaching was realized in the second picoinjection site located downstream from the first in the same microfluidic channel. In this process, monodisperse Hb-alginate-PLL-g-PEG particles with a diameter around the size of RBCs (9 µm) were obtained with around 80% of the 7.5 mg ml-1 Hb included in the injected aqueous alginate retaining in the obtained microparticles. Microparticles with Hb loading (32.8 pg per bead) and retention (28.8 pg per bead) over a week of storage at 4 °C are in accordance with the average amount of Hb per RBC. The Hb-alginate-PLL-g-PEG microbeads fabricated in the size range of RBCs are significant for further exploration.

Interrelation between swelling, mechanical constraints and reaction-diffusion processes in molecular responsive hydrogels.

The net swelling dynamics in molecular responsive hydrogels can be viewed as an integrated effect of discernible processes involving transport of actuating species, reaction with network components like destabilization of physical crosslinks or cleavage of network strands and concomitant network relaxation. Here, we describe a finite element modeling approach coupling these interdependent, underlying processes in hydrogels including oligonucleotide duplexes as physical crosslinks that can be destabilized by a particular molecule. These molecular responsive hydrogels based on acrylamide including either DNA or oligomorpholinos (MO), a DNA analogue, as functional elements can be made with various content of dsDNA or dsMO supported cross-links. The dsDNA or dsMO integrated in the hydrogel can be fabricated with ssDNA designed to competitively displace the connectivity of the dsDNA supported crosslinks, and similar for the MO hydrogels. The overall processes can be framed in a diffusion-reaction scheme. This process is dependent on the concentration of the diffusing species, their diffusion coefficients and their location. Thus, the reaction taking place in particular molecular responsive hydrogels is coupled with the deformations due to swelling and mechanical constraints undergone by the gel. Numerical examples show the importance of coupling reaction-diffusion with mechanical deformations for such gels. Finally, our model is compared to swelling experiments of hemi-spheroidal molecular responsive hydrogels bound to an optical fiber. Parameters of the reaction-diffusion model were obtained by fitting the model to reported experimental data where molecular stimuli designed with different molecular parameters for the competitive displacement reaction were employed in the swelling experiments.

DNA Aptamer Functionalized Hydrogels for Interferometric Fiber-Optic Based Continuous Monitoring of Potassium Ions.

In the present paper, we describe a potassium sensor based on DNA-aptamer functionalized hydrogel, that is capable of continuous label-free potassium ion (K+) monitoring with potential for in situ application. A hydrogel attached to the end of an optical fiber is designed with di-oligonucleotides grafted to the polymer network that may serve as network junctions in addition to the covalent crosslinks. Specific affinity toward K+ is based on exploiting a particular aptamer that exhibits conformational transition from single-stranded DNA to G-quadruplex formed by the di-oligonucleotide in the presence of K+. Integration of this aptamer into the hydrogel transforms the K+ specific conformational transition to a K+ concentration dependent deswelling of the hydrogel. High-resolution interferometry monitors changes in extent of swelling at 1 Hz and 2 nm resolution for the hydrogel matrix of 50 µm. The developed hydrogel-based biosensor displayed high selectivity for K+ ions in the concentration range up to 10 mM, in the presence of physiological concentrations of Na+. Additionally, the concentration dependent and selective K+ detection demonstrated in the artificial blood buffer environment, both at room and physiological temperatures, suggests substantial potential for practical applications such as monitoring of potassium ion concentration in blood levels in intensive care medicine.

A Titratable Cell Lysis-on-Demand System for Droplet-Compartmentalized Ultrahigh-Throughput Screening in Functional Metagenomics and Directed Evolution.

Water-in-oil emulsion droplets are an attractive format for ultrahigh-throughput screening in functional metagenomics and directed evolution applications that allow libraries with more than 107 members to be characterized in a day. Single library members are compartmentalized in droplets that are generated in microfluidic devices and tested for the presence of target biocatalysts. The target proteins can be produced intracellularly, for example, in bacterial hosts in-droplet cell lysis is therefore necessary to allow the enzymes to encounter the substrate to initiate an activity assay. Here, we present a titratable lysis-on-demand (LoD) system enabling the control of the cell lysis rate in Escherichia coli. We demonstrate that the rate of cell lysis can be controlled by adjusting the externally added inducer concentration. This LoD system is evaluated both at the population level (by optical density measurements) and at the single-cell level (on single-cell arrays and in alginate microbeads). Additionally, we validate the LoD system by droplet screening of a phosphotriesterase expressed from E. coli, with cell lysis triggered by inducer concentrations in the µM range. The LoD system yields sufficient release of the intracellularly produced enzymes to bring about a detectable quantity of product (measured by fluorescence in flow cytometry of double emulsions), while leaving viable cells for the downstream recovery of the genetic material.

On the Determination of Mechanical Properties of Aqueous Microgels-Towards High-Throughput Characterization.

Aqueous microgels are distinct entities of soft matter with mechanical signatures that can be different from their macroscopic counterparts due to confinement effects in the preparation, inherently made to consist of more than one domain (Janus particles) or further processing by coating and change in the extent of crosslinking of the core. Motivated by the importance of the mechanical properties of such microgels from a fundamental point, but also related to numerous applications, we provide a perspective on the experimental strategies currently available and emerging tools being explored. Albeit all techniques in principle exploit enforcing stress and observing strain, the realization differs from directly, as, e.g., by atomic force microscope, to less evident in a fluid field combined with imaging by a high-speed camera in high-throughput strategies. Moreover, the accompanying analysis strategies also reflect such differences, and the level of detail that would be preferred for a comprehensive understanding of the microgel mechanical properties are not always implemented. Overall, the perspective is that current technologies have the capacity to provide detailed, nanoscopic mechanical characterization of microgels over an extended size range, to the high-throughput approaches providing distributions over the mechanical signatures, a feature not readily accessible by atomic force microscopy and micropipette aspiration.

Fabrication of monodisperse alginate microgel beads by microfluidic picoinjection: a chelate free approach.

Micron-sized alginate hydrogel beads are extensively employed as an encapsulation medium for biochemical and biomedical applications. Here we report on the microfluidic assisted fabrication of calcium alginate (Ca-alginate) beads by on-chip picoinjection of aqueous calcium chloride (CaCl2) in emulsified aqueous sodium alginate (Na-alginate) droplets or by picoinjection of Na-alginate solution in emulsified aqueous CaCl2 droplets. There is no added chelator to reduce the Ca activity in either of the two strategies. The two fabrication strategies are implemented using a flow-focusing and picoinjection modules in a single PDMS chip. Aqueous alginate solution was emulsified and infused with CaCl2 solution as the squeezed droplet pass the picoinjection channel; consequently, monodisperse, spherical, and structurally homogeneous Ca-alginate beads were obtained. Monodisperse alginate microgel populations with a mean diameter in the range of 8 to 28 µm and standard deviation less than 1 µm were successfully generated using microfluidic channels with various dimensions and controlling the flow parameters. Monodisperse but also non-spherical particles with diameters 6 to 15 µm were also fabricated when picoinjecting Na-alginate solution in emulsified aqueous CaCl2 droplets. The Ca-alginate microbeads fabricated with tailormade size in the range from sub-cellular and upwards were in both strategies realized without any use of chelators or change in pH conditions, which is considered a significant advantage for further exploitation as encapsulation process for improved enzymatic activity and cell viability.

Impact of Silanization Parameters and Antibody Immobilization Strategy on Binding Capacity of Photonic Ring Resonators.

Ring resonator-based biosensors have found widespread application as the transducing principle in "lab-on-a-chip" platforms due to their sensitivity, small size and support for multiplexed sensing. Their sensitivity is, however, not inherently selective towards biomarkers, and surface functionalization of the sensors is key in transforming the sensitivity to be specific for a particular biomarker. There is currently no consensus on process parameters for optimized functionalization of these sensors. Moreover, the procedures are typically optimized on flat silicon oxide substrates as test systems prior to applying the procedure to the actual sensor. Here we present what is, to our knowledge, the first comparison of optimization of silanization on flat silicon oxide substrates to results of protein capture on sensors where all parameters of two conjugation protocols are tested on both platforms. The conjugation protocols differed in the chosen silanization solvents and protein immobilization strategy. The data show that selection of acetic acid as the solvent in the silanization step generally yields a higher protein binding capacity for C-reactive protein (CRP) onto anti-CRP functionalized ring resonator sensors than using ethanol as the solvent. Furthermore, using the BS3 linker resulted in more consistent protein binding capacity across the silanization parameters tested. Overall, the data indicate that selection of parameters in the silanization and immobilization protocols harbor potential for improved biosensor binding capacity and should therefore be included as an essential part of the biosensor development process.

Morpholino Target Molecular Properties Affect the Swelling Process of Oligomorpholino-Functionalized Responsive Hydrogels.

Responsive hydrogels featuring DNA as a functional unit are attracting increasing interest due to combination of versatility and numerous applications. The possibility to use nucleic acid analogues opens for further customization of the hydrogels. In the present work, the commonly employed DNA oligonucleotides in DNA-co-acrylamide responsive hydrogels are replaced by Morpholino oligonucleotides. The uncharged backbone of this nucleic acid analogue makes it less susceptible to possible enzymatic degradation. In this work we address fundamental issues related to key processes in the hydrogel response; such as partitioning of the free oligonucleotides and the strand displacement process. The hydrogels were prepared at the end of optical fibers for interferometric size monitoring and imaged using confocal laser scanning microscopy of the fluorescently labeled free oligonucleotides to observe their apparent diffusion and accumulation within the hydrogels. Morpholino-based hydrogels' response to Morpholino targets was compared to DNA hydrogels' response to DNA targets of the same base-pair sequence. Non-binding targets were observed to be less depleted in Morpholino hydrogels than in DNA hydrogels, due to their electroneutrality, resulting in faster kinetics for Morpholinos. The electroneutrality, however, also led to the total swelling response of the Morpholino hydrogels being smaller than that of DNA, since their lack of charges eliminates swelling resulting from the influx of counter-ions upon oligonucleotide binding. We have shown that employing nucleic acid analogues instead of DNA in hydrogels has a profound effect on the hydrogel response.

Dense carbon-nanotube coating scaffolds stimulate osteogenic differentiation of mesenchymal stem cells.

Carbon nanotubes (CNTs) have desirable mechanical properties for use as biomaterials in orthopedic and dental area such as bone- and tooth- substitutes. Here, we demonstrate that a glass surface densely coated with single-walled carbon nanotubes (SWNTs) stimulate the osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSCs). MSCs incubated on SWNT- and multi-walled carbon nanotube (MWNT)-coated glass showed high activities of alkaline phosphatase that are markers for early stage osteogenic differentiation. Expression of Bmp2, Runx2, and Alpl of MSCs showed high level in the early stage for MSC incubation on SWNT- and MWNT-coated surfaces, but only the cells on the SWNT-coated glass showed high expression levels of Bglap (Osteocalcin). The cells on the SWNT-coated glass also contained the most calcium, and their calcium deposits had long needle-shaped crystals. SWNT coating at high density could be part of a new scaffold for bone regeneration.

Toehold Length of Target ssDNA Affects Its Reaction-Diffusion Behavior in DNA-Responsive DNA-co-Acrylamide Hydrogels.

In the present study, we expand on the understanding of hydrogels with embedded deoxyribonucleic acid (DNA) cross-links, from the overall swelling to characterization of processes that precede the swelling. The hydrogels respond to target DNA strands because of a toehold-mediated strand displacement reaction in which the target strand binds to and opens the dsDNA cross-link. The spatiotemporal evolution of the diffusing target ssDNA was determined using confocal laser scanning microscopy (CLSM). The concentration profiles revealed diverse partitioning of the target DNA inside the hydrogel as compared with the immersing solution: excluding a nonbinding DNA, while accumulating a binding target. The data show that a longer toehold results in faster cross-link opening but reduced diffusion of the target, thus resulting in only a moderate increase in the overall swelling rate. The parameters obtained by fitting the data using a reaction-diffusion model were discussed in view of the molecular parameters of the target ssDNA and hydrogels.

Polysaccharide Hydrogels.

Polysaccharides are a unique source of organic materials in terms of abundance, structural diversity and functionalities[...].

Myeloma-derived extracellular vesicles mediate HGF/c-Met signaling in osteoblast-like cells.

Multiple myeloma is an incurable cancer of antibody-producing plasma cells. Hepatocyte growth factor (HGF), a cytokine aberrantly expressed in half of myeloma patients, is involved in myeloma pathogenesis by enhancing myeloma growth and invasiveness, and may play a role in myeloma bone disease by inhibiting osteoblastogenesis. In this study, we investigated whether extracellular vesicles (EVs) may play a role in HGF signaling between myeloma cells and osteoblast-like target cells. EVs from the HGF-positive cell line JJN-3 and the HGF-negative cell line INA-6, and from bone marrow plasma and primary human myeloma cells, were isolated using sequential centrifugation techniques and the presence of HGF on the EV-surface was investigated with ELISA. EVs from both cell lines were added to an established bioassay where HGF is known to induce interleukin-11 secretion in osteoblast-like cells. Our results show that HGF was bound to the surface of JJN-3-derived EVs, while INA-6-derived EVs were negative for HGF. Only JJN-3-derived EVs induced IL-11 secretion in osteoblast-like recipient cells. When osteoblast-like cells were preincubated with a specific HGF-receptor (c-Met) inhibitor, no induction of interleukin-11 was observed. Downstream c-Met phosphorylation was demonstrated by immunoblotting. EVs isolated from bone marrow plasma and primary myeloma cells were HGF-positive for a subset of myeloma patients. Taken together, this work shows for the first time that HGF bound on the surface of myeloma-derived EVs can effectuate HGF/c-Met signaling in osteoblast-like cells. Myeloma-derived EVs may play a role in myeloma bone disease by induction of the osteoclast-activating cytokine interleukin-11 in osteoblasts.

Nanoparticle-Hydrogel Composites: From Molecular Interactions to Macroscopic Behavior.

Hydrogels are materials used in a variety of applications, ranging from tissue engineering to drug delivery. The incorporation of nanoparticles to yield composite hydrogels has gained substantial momentum over the years since these afford tailor-making and extend material mechanical properties far beyond those achievable through molecular design of the network component. Here, we review different procedures that have been used to integrate nanoparticles into hydrogels; the types of interactions acting between polymers and nanoparticles; and how these underpin the improved mechanical and optical properties of the gels, including the self-healing ability of these composite gels, as well as serving as the basis for future development. In a less explored approach, hydrogels have been used as dispersants of nanomaterials, allowing a larger exposure of the surface of the nanomaterial and thus a better performance in catalytic and sensor applications. Furthermore, the reporting capacity of integrated nanoparticles in hydrogels to assess hydrogel properties, such as equilibrium swelling and elasticity, is highlighted.

Local Structure of Ca2+ Alginate Hydrogels Gelled via Competitive Ligand Exchange and Measured by Small Angle X-Ray Scattering.

Alginates, being linear anionic co-polymers of 1,4-linked residues ß-d-ManA (M) and α-l-GulA (G), are widely applied as hydrogel biomaterials due to their favourable in vivo biocompatibility and convenient ionic crosslinking. The "egg-box" model is the prevailing description of the local structure of junction zones that form between the alginate chains and divalent cations, such as Ca2+, when ionic gelation occurs. In the present study we address to what extent signatures of lateral dimerization and further lateral association of junction zones also represent a valid model for the gelation of alginate using the recently reported method of competitive ligand exchange of chelated Ca2+ ions as a method for introducing gelling ions at constant pH. Small angle X-ray scattering with a q range from 0.1 to 3.3 nm-1 was employed to determine local structure in the hydrogel, using a custom-made fluid sample cell inserted in the X-ray beam. The scattering volume was intended to be localized to the contact zone between the two injected aqueous alginate solutions, and data was captured to resolve the kinetics of the structure formation at three different conditions of pH. The data show evolution of the local structure for the Ca2+ induced formation of junction zones in an alginate with 68% G residues, characterized by cross-sectional radii that could be accounted for by a two-component, broken rod like model. The evolution of the two component weight fractions apparently underpinned the connectivity, as reflected in the rheological data.

Self-Coacervation of a Silk-Like Protein and Its Use As an Adhesive for Cellulosic Materials.

Liquid-liquid phase separation of biomacromolecules plays a critical role in many of their functions, both as cellular components and in structural assembly. Phase separation is also a key mechanism in the assembly of engineered recombinant proteins for the general aim to build new materials with unique structures and properties. Here the phase separation process of an engineered protein with a block-architecture was studied. As a central block, we used a modified spider silk sequence, predicted to be unstructured. In each terminus, folded globular blocks were used. We studied the kinetics and mechanisms of phase formation and analyzed the evolving structures and their viscoelastic properties. Individual droplets were studied with a micropipette technique, showing both how properties vary between individual drops and explaining overall bulk rheological properties. A very low surface energy allowed easy deformation of droplets and led to efficient infiltration into cellulosic fiber networks. Based on these findings, we demonstrated an efficient use of the phase-separated material as an adhesive for cellulose. We also conclude that the condensed state is metastable, showing an ensemble of properties in individual droplets and that an understanding of protein phase behavior will lead to developing a wider use of proteins as structural polymers.

In vitro single-cell dissection revealing the interior structure of cable bacteria.

Filamentous Desulfobulbaceae bacteria were recently discovered as long-range transporters of electrons from sulfide to oxygen in marine sediments. The long-range electron transfer through these cable bacteria has created considerable interests, but it has also raised many questions, such as what structural basis will be required to enable micrometer-sized cells to build into centimeter-long continuous filaments? Here we dissected cable bacteria cells in vitro by atomic force microscopy and further explored the interior, which is normally hidden behind the outer membrane. Using nanoscale topographical and mechanical maps, different types of bacterial cell-cell junctions and strings along the cable length were identified. More important, these strings were found to be continuous along the bacterial cells passing through the cell-cell junctions. This indicates that the strings serve an important function in maintaining integrity of individual cable bacteria cells as a united filament. Furthermore, ridges in the outer membrane are found to envelop the individual strings at cell-cell junctions, and they are proposed to strengthen the junctions. Finally, we propose a model for the division and growth of the cable bacteria, which illustrate the possible structural requirements for the formation of centimeter-length filaments in the recently discovered cable bacteria.

Correction: Interactions between the breast cancer-associated MUC1 mucins and C-type lectin characterized by optical tweezers.

[This corrects the article DOI: 10.1371/journal.pone.0175323.].

Interactions between the breast cancer-associated MUC1 mucins and C-type lectin characterized by optical tweezers.

Carbohydrate-protein interactions govern many crucial processes in biological systems including cell recognition events. We have used the sensitive force probe optical tweezers to quantify the interactions occurring between MGL lectins and MUC1 carrying the cancer-associated glycan antigens mucins Tn and STn. Unbinding forces of 7.6 pN and 7.1 pN were determined for the MUC1(Tn)-MGL and MUC1(STn)-MGL interactions, at a force loading rate of ~40 pN/s. The interaction strength increased with increasing force loading rate, to 27 and 37 pN at a force loading rate of ~ 310 pN/s. No interactions were detected between MGL and MUC1(ST), a glycoform of MUC1 also expressed by breast carcinoma cells. Interestingly, this glycan (ST) can be found on proteins expressed by normal cells, although in this case not on MUC1. Additionally, GalNAc decorated polyethylene glycol displayed similar rupture forces as observed for MUC1(Tn) and MUC1(STn) when forced to unbind from MGL, indicating that GalNAc is an essential group in these interactions. Since the STn glycan decoration is more frequently found on the surface of carcinomas than the Tn glycan, the binding of MUC1 carrying STn to MGL may be more physiologically relevant and may be in part responsible for some of the characteristics of STn expressing tumours.