SNAP‐Tag‐Based Subcellular Protein Labeling and Fluorescent Imaging with Naphthalimides

Genetically encoded technologies provide methods for the specific labeling and imaging of proteins, which is essential to understand the subcellular localization of these proteins and their function. Herein, we employed naphthalimide, an efficient two‐photon fluorophore, to develop O⁶‐benzylguanine (BG) derivatives for specific labeling of subcellular proteins and fluorescent imaging through the SNAP‐tag. Three naphthalimide–BG derivatives, TNI‐BG, QNI‐BG, and ONI‐BG, were conveniently synthesized through modular “click chemistry” in high yields. All of them showed high labeling efficiency with SNAP‐tag in solution (≈1–2×10³ s^?1 m ^?1) and in bacteria. Among them, ONI‐BG showed high specificity to diffused, histone H2B and mitochondria COX8A targeted SNAP‐tag in mammalian cells. The protein‐labeled naphthalimides exhibited high two‐photon absorption cross‐sections, which indicated their potential application in protein‐specific two‐photon fluorescent imaging, such as two‐photon fluorescent lifetime imaging and two‐photon multicolor imaging. Therefore, ONI‐BG is a versatile tool that can be used to track subcellular proteins through multiple fluorescent techniques.     

Synthesis and Metabolic Fate of 4-Methylthiouridine in Bacterial tRNA

Ribonucleic acid (RNA) is central to many life processes and, to fulfill its function, it has a substantial chemical variety in its building blocks. Enzymatic thiolation of uridine introduces 4-thiouridine (s⁴U) into many bacterial transfer RNAs (tRNAs), which is used as a sensor for UV radiation. A similar modified nucleoside, 2-thiocytidine, was recently found to be sulfur-methylated especially in bacteria exposed to antibiotics and simple methylating reagents. Herein, we report the synthesis of 4-methylthiouridine (ms⁴U) and confirm its presence and additional formation under stress in Escherichia coli. We used the synthetic ms⁴U for isotope dilution mass spectrometry and compared its abundance to other reported tRNA damage products. In addition, we applied sophisticated stable-isotope pulse chase studies (NAIL-MS) and showed its AlkB-independent removal in vivo. Our findings reveal the complex nature of bacterial RNA damage repair.     

Phenoxy Radical Reactivity of Nucleic Acids: Practical Implications for Biotinylation

Recent advances in peroxidase-mediated biotin tyramide (BT) signal amplification technology have resulted in high-resolution and subcellular compartment-specific mapping of protein and RNA localization. Horseradish peroxidase (HRP) in the presence of H₂O₂ is known to activate phenolic compounds for phenoxy radical reaction with nucleic acids, where biotinylation by BT is a practical example. BT reactivity with RNA and DNA is not understood in detail. We report that BT phenoxy radicals react in a sequence-independent manner with guanosine bases in RNA. In contrast, DNA reactivity with BT cannot be detected by our methods under the same conditions. Remarkably, we show that fluorescein conjugates DNA rapidly and selectively reacts with BT phenoxy radicals, allowing convenient and practical biotinylation of DNA on fluorescein with retention of fluorescence.     

The Inhibition Effect of Photo-Cross-Linking between Probes in Photo-Induced Double Duplex Invasion DNA

Double duplex invasion (DDI) DNA is a useful antigene method that inhibits expression of genomic DNA. We succeeded in performing photoinduced-DDI (pDDI) using ultrafast photo-cross-linking. 5-Cyanouracil (^CNU) has been used in pDDI to inhibit photo-cross-linking between probes, but its importance has not been clarified. Therefore, in this study, we evaluated the effect of spacer (S) and d-spacer (dS) that exhibit photo-cross-linking ability similar to that of ^CNU. ^CNU exhibited the highest pDDI efficiency, and S, dS, and T were not very different. The photo-cross-linking inhibitory effect was better with S and dS than with thymidine (T). Conversely, the thermal stability was significantly lower with S and dS than with T. The results suggest that the pDDI efficiency is determined by the balance between the photo-cross-linking inhibitory effect and the thermal stability, which is the introduction efficiency for double-stranded DNA. Therefore, ^CNU, which has a photo-cross-linking inhibitory effect and a high T_m value, showed the highest inhibitory efficiency.     

Phospha‐Michael Addition as a New Click Reaction for Protein Functionalization

A new type of click reaction between an alkyl phosphine and acrylamide was developed and applied for site‐specific protein labeling in vitro and in live cells. Acrylamide is a small electrophilic olefin that readily undergoes phospha‐Michael addition with an alkyl phosphine. Our kinetic study indicated a second‐order rate constant of 0.07 m ^?1 s^?1 for the reaction between tris(2‐carboxyethyl)phosphine and acrylamide at pH 7.4. To demonstrate its application in protein functionalization, we used a dansyl–phosphine conjugate to successfully label proteins that were site‐specifically installed with N^?‐acryloyl‐l ‐lysine and employed a biotin–phosphine conjugate to selectively probe human proteins that were metabolically labeled with N‐acryloyl‐galactosamine.     

Fluorescence Modified Chitosan-Coated Magnetic Nanoparticles for High-Efficient Cellular Imaging

Labeling of cells with nanoparticles for living detection is of interest to various biomedical applications. In this study, novel fluorescent/magnetic nanoparticles were prepared and used in high-efficient cellular imaging. The nanoparticles coated with the modified chitosan possessed a magnetic oxide core and a covalently attached fluorescent dye. We evaluated the feasibility and efficiency in labeling cancer cells (SMMC-7721) with the nanoparticles. The nanoparticles exhibited a high affinity to cells, which was demonstrated by flow cytometry and magnetic resonance imaging. The results showed that cell-labeling efficiency of the nanoparticles was dependent on the incubation time and nanoparticles’ concentration. The minimum detected number of labeled cells was around 10⁴ by using a clinical 1.5-T MRI imager. Fluorescence and transmission electron microscopy instruments were used to monitor the localization patterns of the magnetic nanoparticles in cells. These new magneto-fluorescent nanoagents have demonstrated the potential for future medical use.     

Efficient Acid‐Catalyzed 18F/19F Fluoride Exchange of BODIPY Dyes

Fluorine‐containing fluorochromes are important validation agents for positron emission tomography imaging compounds, as they can be readily validated in cells by fluorescence imaging. In particular, the ¹⁸F‐labeled BODIPY‐FL fluorophore has emerged as an important platform, but little is known about alternative ¹⁸F‐labeling strategies or labeling on red‐shifted fluorophores. In this study we explore acid‐catalyzed ¹⁸F/¹⁹F exchange on a range of commercially available N‐hydroxysuccinimidyl ester and maleimide BODIPY fluorophores. We show this method to be a simple and efficient ¹⁸F‐labeling strategy for a diverse span of fluorescent compounds, including a BODIPY‐modified PARP‐1 inhibitor, and amine‐ and thiol‐reactive BODIPY fluorophores.     

Pulse measurements of a large surface photopotential on the type P silicon/electrolyte solution interface

The results of a study on a large pulse surface photopotential of P type silicon electrodes (10 Ω cm) immersed in 0·1 M Na₂SO₄ solutions of various pH are discussed in the paper. The derivative ?(?_s — ?^s)/? pH has been determined and has been found to be ?60 mV in the pH range from 1 to 9. The derivative ?(?_s — ?_b)/? U has been evaluated from theoretical considerations and experimental results to be 0·5 for the studied solutions in the potential range where the dependence of (?_s — ?_b) on U is linear.     

Electro-oxidation of benzene in a fixed bed reactor

A fixed bed electrochemical reactor was used in the laboratory to oxidize benzene to quinone. The reactor consisted of a 3 mm thick bed of 1 mm lead shot, 0.5 m long by 0.05 m wide, sandwiched between a lead feeder plate and an asbestos diaphragm which was compressed against a stainless steel cathode plate. A dispersion of benzene in aqueous sulphuric acid was passed through the reactor and the rates of production of quinone, hydroquinone, carbon dioxide, oxygen and hydrogen, together with the cell voltage and pressure drop, were obtained for a range of operating conditions.Concentrations of quinone in the benzene product varied from 0.04 to 0.31 M and current efficiencies for quinone were in the range 22 to 55%. In a single pass of 1 M acid and benzene through the reactor at 25° C the quinone efficiency fell from 53% to 39% as the average superficial current density increased from 0.4 to 2.0 kA m^–2. At an average superficial current density of 2.0 kA m^–2 the quinone efficiency decreased with an increase in process temperature (25 to 50° C), but increased with increases in acid concentration (1 to 4 M), acid flow (0.5 to 1.0 cm³ s^–1), benzene flow (0.05 to 10 cm³ s^–1) and co-current nitrogen gas flow (0 to 32 cm³ s^–1 at STP). Recycling the 4 M sulphuric acid at 25° C raised the concentration of quinone in the product benzene but decreased the net current efficiency for quinone. Corresponding changes were observed in the cell voltage and in the current efficiencies for hydroquinone, carbon dioxide and oxygen. The results are discussed in terms of the process stoichiometry, electrode kinetics and mass transfer for three-phase flow in a fixed bed reactor.Nomenclature A Acid flow - a ₁ Liquid/liquid specific interfacial area - a _s Liquid/solid specific interfacial area - B Benzene flow - d ₃₂ Sauter mean drop diameter - d _P Particle diameter - E Current efficiency - F Faraday number - I Total current - i _l Superficial transfer-limited current density - K Liquid/liquid distribution coefficient - k _c Liquid/liquid mass transfer coefficient in continuous phase - k _s Liquid/solid mass transfer coefficient - L _c Superficial liquid load — continuous phase - L _d Superficial liquid load — disperse phase - Q _A Quinone concentration in aqueous phase - V ⁰ Standard electrode potential - z Number of electrons per equivalent     

Mass transfer and deodorization efficiency in a countercurrent spray tower for low superficial gas velocities

The aim of this study was to characterize mass transfer and deodorization efficiency in a countercurrent spray tower for low superficial gas velocities. The influence of operating parameters (U_G = 0.005 to 0.025 m s^?1, U_L = 6.1 × 10^?5 to 2.4 × 10^?4 m s^?1) on the liquid retention (ε_L), the drop diameter (d_g), the interfacial area (a) and the overall liquid and gas phase mass transfer coefficients (K_La, K_Ga) were estimated. The spray efficiency of some malodorous compounds was also estimated. A negative influence of the superficial gas velocity was demonstrated, during the spraying of water or chemical neutralizing scrubbing solutions. There was also an increase with the liquid flow rate. Abatements obtained were very good with respect to ammonia (>90%), and acceptable for the other compounds.     

Plasmonic organic bulk–heterojunction solar cells based on hydrophobic gold nanorod insertion into active layers

In this work, we fabricated plasmonic organic bulk–heterojunction solar cells by inserting hydrophobic gold nanorods (GNRs) into the active layers. Power conversion efficiency was improved from 7.43% to 8.22% because the plasmonic effect of GNRs improved the light harvesting efficiency. Maximum exciton generation rate was increased from 1.35 × 10⁻²⁶ to 1.51× 10⁻²⁶m⁻³ s⁻¹, and the electron mobility was also increased from 8.6 × 10⁻⁵ to 1.5× 10⁻⁴cm⁻² V⁻¹ s⁻¹. As a result, the short circuit current density was improved from 15.5 to 16.7 mA cm⁻²—the dominant reason for performance enhancement. The open circuit voltage and fill factor were improved simultaneously. The plasmonic device showed a highest PCE of 8.43%, indicating that doping GNRs into active layers is a simple and effective way to fabricate high‐performance organic solar cells. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 45920.     

Arylamine polymers prepared via facile paraldehyde addition condensation: an effective hole‐transporting material for perovskite solar cells

Arylamine polymers were prepared via the facile one‐step addition condensation of N,N′‐diphenyl‐N,N′‐bis(4‐methylphenyl)‐1,4‐phenylenediamine and 4‐methoxytriphenylamine with paraldehyde. The polymers were highly soluble in common organic solvents. The non‐conjugated arylamine polymer structure was characterized and found to form tough, homogeneous, amorphous layers with a glass transition temperature above 200 °C on a substrate by a simple spin‐coating process. The polymer layers exhibited a hole mobility of the order of 10^?5 cm² V^?1 s^?1, which was comparable with those of previously reported arylamine polymers, and a highest occupied molecular orbital level of ?5.38 eV appropriate for the hole‐transporting layer of perovskite solar cells. The perovskite cells fabricated with the polymers gave a photovoltaic conversion efficiency of 16.0%. © 2018 Society of Chemical Industry     

Green-Emitting Rhodamine Dyes for Vital Labeling of Cell Organelles Using STED Super-Resolution Microscopy

Fluorescence microscopy reveals the localization, spatial distribution, and temporal dynamics of the specifically labeled organelles in living cells. Labeling with exogenous conjugates prepared from fluorescent dyes and small molecules (ligands) is an attractive alternative to the use of fluorescent proteins, but proved to be challenging due to insufficient cell-permeability of the probes, unspecific staining, or low dye brightness. We evaluated four green-emitting rhodamine dyes and their conjugates intended for the specific labeling of lysosomes, mitochondria, tubulin, and actin in living cells. The imaging performance of the probes in living human fibroblasts has been studied by using confocal and stimulated emission depletion (STED) super-resolution microscopy with a commercial 595 nm STED laser. Two bright and photostable dyes (LIVE 510 and LIVE 515) provide specific and versatile staining.     

MULTINOMIAL SUBSET SELECTION USING INVERSE SAMPLING AND ITS EFFICIENCY WITH RESPECT TO FIXED SAMPLING

The multinomial selection problem is considered in its general form where the objective is to select a subset of s cells which contain the t ‘best’ cells, s ≥ t. The inverse-sampling procedure is studied for this problem and the LFC is derived under the difference zone. An expression for the relative efficiency of this procedure with respect to the widely used fixed-sample-size selection procedure is obtained and theoretical bounds are derived for this efficiency. It is found that the inverse-sampling procedure performs uniformly better than the usual fixed-sampling procedure in the case s = t and is often more efficient for s > t. When the selection goal is to select any c of the t best cells, using a subset of s cells, expressions for efficiency may be similarly obtained.     

Vicinal Diol-Tethered Nucleobases as Targets for DNA Redox Labeling with Osmate Complexes

Six-valent osmium (osmate) complexes with nitrogenous ligands have previously been used for the modification and redox labeling of biomolecules involving vicinal diol moieties (typically, saccharides or RNA). In this work, aliphatic (3,4-dihydroxybutyl and 3,4-dihydroxybut-1-ynyl) or cyclic (6-oxo-6-(cis-3,4-dihydroxypyrrolidin-1-yl)hex-2-yn-1-yl, PDI) vicinal diols are attached to nucleobases to functionalize DNA for subsequent redox labeling with osmium(VI) complexes. The diol-linked 2′-deoxyribonucleoside triphosphates were used for the polymerase synthesis of diol-linked DNA, which, upon treatment with K₂OsO₃ and bidentate nitrogen ligands, gave the desired Os-labeled DNA, which were characterized by means of the gel-shift assay and ESI-MS. Through ex situ square-wave voltammetry at a basal plane pyrolytic graphite electrode, the efficiency of modification/labeling of individual diols was evaluated. The results show that the cyclic cis-diol (PDI) was a better target for osmylation than that of the flexible aliphatic ones (alkyl- or alkynyl-linked). The osmate adduct-specific voltammetric signal obtained for Os^VI-treated DNA decorated with PDI showed good proportionality to the number of PDI per DNA molecule. The Os^VI reagents (unlike OsO₄) do not attack nucleobases; thus offering specificity of modification on the introduced glycol targets.     

Mass transfer to and copper deposition on a round bar in a new type of electrolytic cell: the helix cell

High-rate electrodeposition of copper from CuSO₄-H₂SO₄ baths can be achieved by using crossflow of solution. To obtain copper layers of uniform thickness and quality, a new type of electrolytic cell, the helix cell, has been proposed. An experimental dimensionless relation has been given to describe the mass transfer to a round bar, in crossflow, in a helix cell. Moreover, the current efficiency of copper deposition has been obtained as a function of current density, flow rate of solution, temperature and weight per cent CuSO₄ in the CuSO₄-H₂SO₄ solution.Nomenclature A _e working-electrode surface area (m²) - c concentration of Cu²⁺ (mol m^-3) - c _e c at electrode surface (mol m^-3) - c _b c in bulk of solution (mol m^-3) - C constant factor (-) - d _c inner diameter of central cylinder of helix cell (mm) - d _c volumetric hydraulic diameter of helix cell (mm) - d _h width of helical slots in central cylinder of helix cell (mm) - d _s diameter of working electrode (round bar) (mm) - D _i diffusion coefficient of species i (m² s^-1) - F Faraday constant (C mol^-1) - I current (A) - k _i mass-transfer coefficient for a species i (m s^-1) - i current density (kA m^-2, A m^-2) - L _c length of working-electrode compartment of helix cell (m) - n number of electrons involved in electrode reaction (-) - n₁, n₂, n₃ constants (-) - R gas constant: R=8.31 J K^-1 mol^-1 - Re Reynolds number: Re=v _c d _h/v (-) - Sc Schmidt number: Sc=v/D (-) - Sh Sherwood number: Sh=kd _h/D - t time (s) - T temperature (K) - U _s volumetric rate of solution through the helix cell (m³ s^-1) - v _c flow rate of solution through working-electrode compartment of the helix cell (vc = U/(d _c –dw)Lc) (ms–1) - density of solution (kg m ^-3) - u dynamic viscosityofsolution (kg m-1 s-1) - v kinematic viscosity of solution (m2 s-1) - _i current efficiency for formation of a species i (-)     

A Quencher-Free Linear Probe from Serinol Nucleic Acid with a Fluorescent Uracil Analogue

With the goal of developing a quencher-free probe composed of an artificial nucleic acid, the fluorescent nucleobase analogue 5-(perylenylethynyl)uracil (^PeU), which was incorporated into totally artificial serinol nucleic acid (SNA) as a substitute for thymine, has been synthesized. In the context of a 12-mer duplex with RNA, these fluorophores reduce duplex stability slightly compared with that of an SNA without ^PeU modification; thus suggesting that structural distortion is not induced by the modification. If two ^PeUs were incorporated at separate positions in an SNA, the fluorescent emission at λ≈490 nm was clearly enhanced upon hybridization with complementary RNA. A quencher-free SNA linear probe containing three ^PeUs, each separated by six nucleobases, has been designed. Detection of target RNA with high sensitivity and discrimination of a single-base mismatch has also been demonstrated.     

Ceric ion‐induced graft copolymerization of acrylamide on cationic guar gum at low temperature

The solution polymerization of acrylamide (AM) on cationic guar gum (CGG) under nitrogen atmosphere using ceric ammonium sulfate (CAS) as the initiator has been realized. The effects of monomer concentration and reaction temperature on grafting conversion, grafting ratio, and grafting efficiency (GE) have been studied. The optimal conditions such as 1.3 mol of AM monomer and 2.2 × 10^?4 mol of CAS have been adopted to produce grafted copolymer (CGG1‐g‐PAM) of high GE of more than 95% at 10°C. The rates of polymerization (R_p) and rates of graft copolymerization (R_g) are enhanced with increase in temperature (<35°C).The R_p is enhanced from 0.43 × 10^?4 mol L^?1 s^?1 for GG‐g‐PAM to 2.53 × 10^?4 mol L^?1 s^?1 for CGG1‐g‐PAM (CGG1, degree of substitute (DS) = 0.007), and R_g from 0.42 × 10^?4 to 2.00 × 10^?4 mol L^?1 s^?1 at 10°C. The apparent activation energy is decreased from 32.27 kJ mol^?1 for GG‐g‐PAM to 8.09 kJ mol^?1 for CGG1‐g‐PAM, which indicates CGG has higher reactivity than unmodified GG ranging from 10 to 50°C. Increase of DS of CGG will lead to slow improvement of the polymerization rates and a hypothetical mechanism is put forward. The grafted copolymer has been characterized by infrared spectroscopy, thermal analysis, and scanning electron microscopy. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 3715–3722, 2007     

Pressure drop and separation efficiency in a flooded hydrocyclone

The pressure drop and separation efficiency were measured of a hydrocyclone operating under flooded underflow or no air-core conditions. The solids were glass spheres with a geometric number average diameter of 13 μm and a geometric standard deviation of 1.69. Water was the fluid. Other fixed variables included the solids feed concentration of 2265 mg/L, the glass hydrocarbon configuration following the optimal Rietema recommendations and the temperature. The variables were feed flowrate 0.44 to 0.63 L.s¹ and volume split of 2.4:1 to 6.1:1; a factorial design was used. The pressure drop, ΔP_s, was 4 to 7 times larger than that expected for air core operation and was correlated in terms of the capacity ratio Q/(ΔP_s)^0.47. The pressure drop was relatively independent of volume split. The separation efficiency was a much stronger function of volume split than of feed flowrate. The D_p(50) diameter at a feed flowrate of 0.63 L.s⁻¹ lies between 4.3 to 9.2 μm; of the seven correlations available for predicting D_p(50), the approach of Rietema (1961) gives the best results. The experimentally measured reduced efficiency was less sharp than that reported by Yoshioka and Hotta (1955) and by the theory of Uematu et al. (1962).