Engineered Methionine Adenosyltransferase Cascades for Metabolic Labeling of Individual DNA Methylomes in Live Cells.

Methylation, a widely occurring natural modification serving diverse regulatory and structural functions, is carried out by a myriad of S-adenosyl-l-methionine (AdoMet)-dependent methyltransferases (MTases). The AdoMet cofactor is produced from l-methionine (Met) and ATP by a family of multimeric methionine adenosyltransferases (MAT). To advance mechanistic and functional studies, strategies for repurposing the MAT and MTase reactions to accept extended versions of the transferable group from the corresponding precursors have been exploited. Here, we used structure-guided engineering of mouse MAT2A to enable biocatalytic production of an extended AdoMet analogue, Ado-6-azide, from a synthetic methionine analogue, S-(6-azidohex-2-ynyl)-l-homocysteine (N3-Met). Three engineered MAT2A variants showed catalytic proficiency with the extended analogues and supported DNA derivatization in cascade reactions with M.TaqI and an engineered variant of mouse DNMT1 both in the absence and presence of competing Met. We then installed two of the engineered variants as MAT2A-DNMT1 cascades in mouse embryonic stem cells by using CRISPR-Cas genome editing. The resulting cell lines maintained normal viability and DNA methylation levels and showed Dnmt1-dependent DNA modification with extended azide tags upon exposure to N3-Met in the presence of physiological levels of Met. This for the first time demonstrates a genetically stable system for biosynthetic production of an extended AdoMet analogue, which enables mild metabolic labeling of a DNMT-specific methylome in live mammalian cells.

Diblock Copolymers of Methacryloyloxyethyl Phosphorylcholine and Dopamine Methacrylamide: Synthesis and Real-Time Adsorption Dynamics by SEIRAS and RAIRS.

Amphiphilic diblock copolymers containing a block of 2-methacryloyloxyethyl phosphorylcholine (MPC) with unique properties to prevent nonspecific protein adsorption and enhance lubrication in aqueous media and a block of dopamine methacrylamide (DOPMA) distinguished by excellent adhesion performance were synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization for the first time. The DOPMA monomer with an acetonide-protected catechol group (acetonide-protected dopamine methacrylamide (ADOPMA)) was used, allowing the prevention of undesirable side reactions during polymerization and oxidation during storage. The adsorption behavior of the diblock copolymers with protected and unprotected catechol groups on gold surfaces was probed using attenuated total reflection (ATR)-Fourier transform infrared (FTIR) spectroscopy, surface-enhanced infrared absorption spectroscopy (SEIRAS), and reflection-absorption infrared spectroscopy (RAIRS). The copolymers pMPC-b-pADOPMA demonstrated physisorption with rapid adsorption and ultrasound-assisted desorption, while the copolymers pMPC-b-DOPMA exhibited chemical adsorption with slower dynamics but a stronger interaction with the gold surface. SEIRAS and RAIRS allowed proving the reorientation of the diblock copolymers during adsorption, demonstrating the exposure of the pMPC block toward the aqueous phase.

Biocompatible Upconverting Nanoprobes for Dual-Modal Imaging and Temperature Sensing.

The demand for multimodal nanomaterials has intensified in recent years driven by the need for ultrasensitive bioimaging probes and accurate temperature monitoring in biological objects. Among the different multimodal nanomaterials that have been extensively studied in the past decade, upconverting nanoparticles are among the most promising. In this paper, we report the synthesis of upconverting nanoparticles with complex core-shell compositions, capable of being excited by 808 or 980 nm laser irradiation and exhibiting a good MRI response. The synthesized nanoparticles also demonstrated high colloidal stability in both aqueous and biological media as well as temperature-sensing capabilities, including the physiological range. Furthermore, the upconversion nanoparticles exhibited significantly lower cytotoxicity for HEK293T cells than the commercially available MRI contrast agent Gd-DTPA.

Real-time observation of ion exchange dynamics during surface treatment of all-inorganic perovskite quantum dots with Zn-halogenide complexes for color tuning and enhanced quantum efficiency.

All-inorganic lead perovskite quantum dots (QDs), due to their distinctive optical properties, have become one of the "hottest" topics in materials science; therefore, the development of new QD synthesis methods or their emission color adjustment is of great interest. Within this study, we present the simple preparation of QDs employing a novel ultrasound-induced hot-injection method, which significantly reduces the QD synthesis time from several hours to merely 15-20 minutes. Moreover, the post-synthesis treatment of perovskite QDs in solutions using zinc halogenide complexes could increase the QD emission intensity and, at the same time, boost their quantum efficiency. This behavior is due to the zinc halogenide complex's ability to remove or significantly reduce the number of surface electron traps in perovskite QDs. Finally, the experiment that shows the ability to instantly adjust the desired emission color of perovskite QDs by variation of the amount of added zinc halogenide complex is presented. The instantly obtained perovskite QD colors cover virtually the full range of the visible spectrum. The zinc halogenide modified perovskite QDs exhibit up to 10-15% higher QEs than those prepared by an individual synthesis.

Biosynthesis of Silver Nanoparticles Produced Using Geobacillus spp. Bacteria.

Silver nanoparticles (AgNPs) are well known for their unique physical and chemical properties, which can be incorporated into a wide range of applications. The growing resistance of microorganisms to antimicrobial compounds promoted the use of AgNPs in antimicrobial therapy. AgNPs can be obtained using physical and chemical methods, but these technologies are highly unfriendly to nature and produce large amounts of side compounds (for example, sodium borohydride and N,N-dimethylformamide). Therefore, alternative technologies are required for obtaining AgNPs. This report focuses on the biosynthesis of silver nanoparticles through the reduction of Ag+ with the cell-free secretomes of four Geobacillus bacterial strains, namely, 18, 25, 95, and 612. Only a few studies that involved Geobacillus bacteria in the synthesis of metal nanoparticles, including AgNPs, have been reported to date. The silver nanoparticles synthesized through bio-based methods were characterized using UV-Vis spectroscopy, scanning electron microscopy (SEM), dynamic light scattering (DLS), and zeta potential measurements. UV-Vis spectroscopy showed a characteristic absorbance peak at 410-425 nm, indicative of AgNPs. SEM analysis confirmed that most nanoparticles were spherical. DLS analysis showed that the sizes of the obtained AgNPs were widely distributed, with the majority less than 100 nm in diameter, while the zeta potential values ranged from -25.7 to -31.3 mV and depended on the Geobacillus spp. strain.

Synthesis and Study of Thermoresponsive Amphiphilic Copolymers via RAFT Polymerization.

Synthesis and study of well-defined thermoresponsive amphiphilic copolymers with various compositions were reported. Kinetics of the reversible addition-fragmentation chain transfer (RAFT) (co)polymerization of styrene (St) and oligo(ethylene glycol) methyl ether methacrylate (PEO5MEMA) was studied by size exclusion chromatography (SEC) and 1H NMR spectroscopy, which allows calculating not only (co)polymerization parameters but also gives valuable information on RAFT (co)polymerization kinetics, process control, and chain propagation. Molecular weight Mn and dispersity D of the copolymers were determined by SEC with triple detection. The detailed investigation of styrene and PEO5MEMA (co)polymerization showed that both monomers prefer cross-polymerization due to their low reactivity ratios (r1 < 1, r2 < 1); therefore, the distribution of monomeric units across the copolymer chain of p(St-co-PEO5MEMA) with various compositions is almost ideally statistical or azeotropic. The thermoresponsive properties of p(St-co-PEO5MEMA) copolymers in aqueous solutions as a function of different hydrophilic/hydrophobic substituent ratios were evaluated by measuring the changes in hydrodynamic parameters under applied temperature using the dynamic light scattering method (DLS).

Polymer brush coated upconverting nanoparticles with improved colloidal stability and cellular labeling.

Upconverting nanoparticles (UCNPs) possess great potential for biomedical application. UCNPs absorb and convert near-infrared (NIR) radiation in the biological imaging window to visible (Vis) and even ultraviolet (UV) radiation. NIR excitation offers reduced scattering and diminished autofluorescence in biological samples, whereas the emitted UV-Vis and NIR photons can be used for cancer treatment and imaging, respectively. However, UCNPs are usually synthesized in organic solvents and are not readily suitable for biomedical application due to the hydrophobic nature of their surface. Herein, we have removed the hydrophobic ligands from the synthesized UCNPs and coated the bare UCNPs with two custom-made hydrophilic polyelectrolytes (synthesized via the reversible addition-fragmentation chain transfer (RAFT) polymerization method). Polymers containing different amounts of PEGylated and carboxylic groups were studied. Coating with both polymers increased the upconversion (UC) emission intensity and photoluminescence lifetime values of the UCNPs, which directly translates to more efficient cancer cell labeling nanoprobes. The polymer composition plays a crucial role in the modification of UCNPs, not only with respect to their colloidal stability, but also with respect to the cellular uptake. Colloidally unstable bare UCNPs aggregate in cell culture media and precipitate, rendering themselves unsuitable for any biomedical use. However, stabilization with polymers prevents UCNPs from aggregation, increases their uptake in cells, and improves the quality of cellular labeling. This investigation sheds light on the appropriate coating for UCNPs and provides relevant insights for the rational development of imaging and therapeutic tools.

Solid-State NMR and Impedance Spectroscopy Study of Spin Dynamics in Proton-Conducting Polymers: An Application of Anisotropic Relaxing Model.

The 1H-13C cross-polarization (CP) kinetics in poly[2-(methacryloyloxy)ethyltrimethylammonium chloride] (PMETAC) was studied under moderate (10 kHz) magic-angle spinning (MAS). To elucidate the role of adsorbed water in spin diffusion and proton conductivity, PMETAC was degassed under vacuum. The CP MAS results were processed by applying the anisotropic Naito and McDowell spin dynamics model, which includes the complete scheme of the rotating frame spin-lattice relaxation pathways. Some earlier studied proton-conducting and nonconducting polymers were added to the analysis in order to prove the capability of the used approach and to get more general conclusions. The spin-diffusion rate constant, which describes the damping of the coherences, was found to be strongly depending on the dipolar I-S coupling constant (DIS). The spin diffusion, associated with the incoherent thermal equilibration with the bath, was found to be most probably independent of DIS. It was deduced that the drying scarcely influences the spin-diffusion rates; however, it significantly (1 order of magnitude) reduces the rotating frame spin-lattice relaxation times. The drying causes the polymer hardening that reflects the changes of the local order parameters. The impedance spectroscopy was applied to study proton conductivity. The activation energies for dielectric relaxation and proton conductivity were determined, and the vehicle-type conductivity mechanism was accepted. The spin-diffusion processes occur on the microsecond scale and are one order faster than the dielectric relaxation. The possibility to determine the proton location in the H-bonded structures in powders using CP MAS technique is discussed.

Effect of Cationic Brush-Type Copolymers on the Colloidal Stability of GdPO4 Particles with Different Morphologies in Biological Aqueous Media.

In this study, we present the synthesis of cationic brush-type polyelectrolytes and their use in the stabilization of GdPO4 particles in aqueous media. Polymers of various compositions were synthesized via the RAFT polymerization route. SEC equipped with triple detection (RI, DP, RALS, and LALS) was used to determine the molecular parameters (Mn, Mw, Mw/Mn). The exact composition of synthesized polymers was determined using NMR spectroscopy. Cationic brush-type polymers were used to improve the stability of aqueous GdPO4 particle dispersions. First, the IEPs of GdPO4 particles with different morphologies (nanorods, hexagonal nanoprisms, and submicrospheres) were determined by measuring the zeta potential of bare particle dispersions at various pH values. Afterward, cationic brush-type polyelectrolytes with different compositions were used for the surface modification of GdPO4 particles (negatively charged in alkaline media under a pH value of ∼10.6). The concentration and composition effects of used polymers on the change in particle surface potential and stability (DLS measurements) in dispersions were investigated and presented in this work. The most remarkable result of this study is redispersible GdPO4 nanoparticle colloids with increased biocompatibility and stability as well as new insights into possible cationic brush-type polyelectrolyte applicability in both scientific and commercial fields.

Ultralight Magnetic Nanofibrous GdPO4 Aerogel.

Anisotropic aerogels are promising bulk materials with a porous 3D structure, best known for their large surface area, low density, and extremely low thermal conductivity. Herein, we report the synthesis and some properties of ultralight magnetic nanofibrous GdPO4 aerogels. Our proposed GdPO4 aerogel synthesis route is eco-friendly and does not require any harsh precursors or conditions. The most common route for magnetic aerogel preparation is the introduction of magnetic nanoparticles into the structure during the synthesis procedure. However, the nanofibrous GdPO4 aerogel reported in this work is magnetic by itself already and no additives are required. The hydrogel used for nanofibrous GdPO4 aerogel preparation was synthesized via a hydrothermal route. The hydrogel was freeze-dried and heat-treated to induce a phase transformation from the nonmagnetic trigonal to magnetic monoclinic phase. Density of the obtained magnetic nanofibrous monoclinic GdPO4 aerogel is only ca. 8 mg/cm3.

CP MAS kinetics in soft matter: Spin diffusion, local disorder and thermal equilibration in poly(2-hydroxyethyl methacrylate).

The 1H-13C cross-polarization magic angle spinning kinetics was studied in poly(2-hydroxyethyl methacrylate) (pHEMA), i.e. a soft material with high degrees of internal freedom and molecular disorder, having the purpose to track the influence of increasing local incoherent contributions to the effects of coherent nature in the open quantum spin systems. The experimental CP MAS kinetic curves were analyzed in the frame of the models of isotropic and anisotropic spin diffusion with thermal equilibration. The rates of spin diffusion and spin-lattice relaxation as well as the profiles of distribution of dipolar coupling, the parameters accounting the effective size of spin clusters and the local order parameters were determined. The intensities of the peaks of periodic quasi-equilibrium origin gradually decrease with increasing disorder, i.e. going from most ordered to more disordered sites in the polymer. Assuming that the thermal motion induced by the temperature gradients is much faster than the equilibration driven by spin diffusion due the difference in spin temperatures, it was deduced that the thermal equilibration in pHEMA occurs in the time scale of 10-4 s. This is one order of magnitude faster than the spectral spin diffusion, which occurs between spins having different resonance frequencies. The thermal equilibration in the case of remote spin clusters was described by the 'stretched exponent' decay. This led to the fractal dimension Dp ≈ 1.65 for both carbon sites (quaternary and carbonyl). The obtained Dp value corresponds to the aggregates, which images are very similar to those for pHEMA and some other related polymer structures are usually conceived.

Bioinspired Adhesion Polymers: Wear Resistance of Adsorption Layers.

Mussel adhesive polymers owe their ability to strongly bind to a large variety of surfaces under water to their high content of 3,4-dihydroxy-l-phenylalanine (DOPA) groups and high positive charge. In this work, we use a set of statistical copolymers that contain medium-length poly(ethylene oxide) side chains that are anchored to the surface in three different ways: by means of (i) electrostatic forces, (ii) catechol groups (as in DOPA), and (iii) the combination of electrostatic forces and catechol groups. A nanotribological scanning probe method was utilized to evaluate the wear resistance of the formed layers as a function of normal load. It was found that the combined measurement of surface topography and stiffness provided an accurate assessment of the wear resistance of such thin layers. In particular, surface stiffness maps allowed us to identify the initiation of wear before a clear topographical wear scar was developed. Our data demonstrate that the molecular and abrasive wear resistance on silica surfaces depends on the anchoring mode and follows the order catechol groups combined with electrostatic forces > catechol groups alone > electrostatic forces alone. The devised methodology should be generally applicable for evaluating wear resistance or "robustness" of thin adsorbed layers on a variety of surfaces.

Effect of structure of cationic comb copolymers on their adsorption and stabilization of titania nanoparticles.

Cationic linear polymer poly([2-(methacryloyloxy)ethyl] trimethylammonium chloride) p(METAC), neutral brush polymer poly(poly(ethylene glycol) methyl ether methacrylate) p(PEO22MEMA), and cationic comb copolymers p(METAC-PEO(x)MEMA) were used for the stabilization of titania dispersions under neutral and alkaline conditions. Random comb copolymers p(METAC-PEO(x)MEMA) differing in charge density and length of PEO side chains were synthesized by RAFT. The adsorption of cationic polymers on titania nanoparticles was evaluated by thermogravimetric analysis; changes in surface potential, by measuring the zeta potential; and the stability of the treated TiO2 dispersions, by laser diffraction and DLS. Cationic linear and comb copolymers containing relatively short PEO side chains promoted the inversion of nanoparticle surface potential from strongly negative (-60 mV) to moderately positive (10-35 mV). Cationic comb copolymers containing longer PEO side chains increased the zeta potential of the treated nanoparticles but did not invert it to positive. Aqueous dispersions of titania nanoparticles stabilized by cationic comb copolymers under alkaline conditions (pH 10) were dispersed by high-energy planetary ball milling up to a primary particle size of 20 nm and were stable for at least 2 days.