Photoredox-Catalyzed Labeling of Hydroxyindoles with Chemoselectivity (PhotoCLIC) for Site-Specific Protein Bioconjugation

We have developed a novel visible-light-catalyzed bioconjugation reaction, PhotoCLIC, that enables chemoselective attachment of diverse aromatic amine reagents onto a site-specifically installed 5-hydroxytryptophan residue (5HTP) on full-length proteins of varied complexity. The reaction uses catalytic amounts of methylene blue and blue/red light-emitting diodes (455/650 nm) for rapid site-specific protein bioconjugation. Characterization of the PhotoCLIC product reveals a unique structure formed likely through a singlet oxygen-dependent modification of 5HTP. PhotoCLIC has a wide substrate scope and its compatibility with strain-promoted azide-alkyne click reaction, enables site-specific dual-labeling of a target protein.     

Zur Bestimmung von Kohlendioxyd in Carbonaten

Ohne Zusammenfassung     

Self-assembled films of cellulose nanofibrils and poly(<Emphasis Type="Italic">o</Emphasis>-ethoxyaniline)

Nanostructured films of poly(o-ethoxyaniline) (POEA) alternated with cellulose nanofibrils (CnF) were successfully produced by self assembly (SA) at different pH values and investigated by atomic force microscopy and ultraviolet-visible spectroscopy. Results show that it was possible to build up films by alternating POEA and CnF layers with relatively precise architectural control by controlling the number of layers and pH. Film thickness had a dependence on pH which is a combination of the effects of the deposited amount for each POEA layer and the pH at which the absorption of the cellulose nanofibrils was carried out. Comparison of alternated layers of POEA and CnF with multi-immersions of POEA at different pH values, as measured by the ratio between slopes of the straight lines of deposited amount of polymer versus the number of self-assembled layers, shows that alternate deposition at pH 2 has a fourfold increase in the slope. Alternatively, at pH 5, there is no significant difference whether the deposition is alternated (POEA–CnF) or not (POEA).     

Surface characterization of untreated and solvent-extracted rice straw

Surface characteristics of untreated and solvent extracted rice straw and intact rice plant stem and sheath tissue have been investigated using reflectance Fourier transform infrared spectroscopy (FTIR-ATR), electrokinetic analysis (EKA), and scanning electron microscopy (SEM). Klason lignin and Kappa numbers were determined according to TAPPI standard methods. Sohxlet extractions were performed with hexanes, ethyl acetate, chloroform, ethanol-toluene and benzene-ethanol azeotropes. The FTIR reflectance spectra showed differences between the untreated sheath and stem tissues. It was found that the functional groups of the stem interior tissues were masked by another substance, which is proposed to be potassium oxalate monohydrate. This mineral salt, readily removed by water-washing, was affected by the solvent extractions with varying results. The extracted amounts ranged from 1 to 7%. While lipid removal was detectable by the FTIR reflectance method, the streaming potential method showed only slight differences between the curves representing the zeta potential (mV) as a function of pH of the untreated versus extracted samples. Reduced values of the iso-electric points were observed for the extracted samples as compared to the untreated rice straw, indicative of more acidic surface characteristics.     

Synthesis of macrocyclic conjugated ψ-disacharides via nitrile oxide cycloaddition: Alkenyl chain length controlled unprecedented formation of di-oxa heterocycles from monosaccharides

Various symmetrical and unsymmetrical isoxazoline embodied chiral macroheterocycles were synthesized from carbohydrate derived oximes. Our result shows that the mode of cycloaddition is dependent on the length and tether present in alkenyl chain of sugar moiety. Thus in some cases inter- and intramolecular cycloaddition of nitrile oxide were observed with high selectivity. The cycloadducts contains two sugar units linked via macrocycles thus they are considered as pseudo-disacharides. The stereochemistry of the newly formed chiral centers was established by 2D-NMR techniques and X-ray crystallographic studies.     

Redox Reactions of Biologically Active Molecules upon Cold Atmospheric Pressure Plasma Treatment of Aqueous Solutions

Cold atmospheric pressure plasma (CAPP) is widely used in medicine for the treatment of diseases and disinfection of bio-tissues due to its antibacterial, antiviral, and antifungal properties. In agriculture, CAPP accelerates the imbibition and germination of seeds and significantly increases plant productivity. Plasma is also used to fix molecular nitrogen. CAPP can produce reactive oxygen and nitrogen species (RONS). Plasma treatment of bio-tissue can lead to numerous side effects such as lipid peroxidation, genotoxic problems, and DNA damage. The mechanisms of occurring side effects when treating various organisms with cold plasma are unknown since RONS, UV-Vis light, and multicomponent biological tissues are simultaneously involved in a heterogeneous environment. Here, we found that CAPP can induce in vitro oxidation of the most common water-soluble redox compounds in living cells such as NADH, NADPH, and vitamin C at interfaces between air, CAPP, and water. CAPP is not capable of reducing NAD⁺ and 1,4-benzoquinone, despite the presence of free electrons in CAPP. Prolonged plasma treatment of aqueous solutions of vitamin C, 1,4-hydroquinone, and 1,4-benzoquinone respectively, leads to their decomposition. Studies of the mechanisms in plasma-induced processes can help to prevent side effects in medicine, agriculture, and food disinfection.     

An Efficient Opal-Suppressor Tryptophanyl Pair Creates New Routes for Simultaneously Incorporating up to Three Distinct Noncanonical Amino Acids into Proteins in Mammalian Cells**

Site-specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into proteins in mammalian cells is a promising technology, where each ncAA must be assigned to a different orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pair that reads a distinct nonsense codon. Available pairs suppress TGA or TAA codons at a considerably lower efficiency than TAG, limiting the scope of this technology. Here we show that the E. coli tryptophanyl (EcTrp) pair is an excellent TGA-suppressor in mammalian cells, which can be combined with the three other established pairs to develop three new routes for dual-ncAA incorporation. Using these platforms, we site-specifically incorporated two different bioconjugation handles into an antibody with excellent efficiency, and subsequently labeled it with two distinct cytotoxic payloads. Additionally, we combined the EcTrp pair with other pairs to site-specifically incorporate three distinct ncAAs into a reporter protein in mammalian cells.     

Experimental and theoretical study of the atmospherically important O2-H2O complex

The theoretically predicted water-oxygen van der Waals adduct has been experimentally confirmed by vibrational characterization using matrix isolation spectroscopic studies at 10 K. Vibrational bands for asymmetric and symmetric OH-stretching for this adduct have been found at 3728 cm(-1) and 3639 cm(-1), respectively. Theoretical calculations performed with Gaussian 98 software at the MP2/6-311++G(2d,2p) level of theory support the alternative structure of the hydrated complex proposed by this study.     

Electrospinning of Polyaniline/Poly(Lactic Acid) Ultrathin Fibers: Process and Statistical Modeling using a Non‐Gaussian Approach

Fibers of poly(lactic acid) (PLA) blended with p‐toluenesulfonic acid‐doped polyaniline, PAni.TSA, were obtained by electrospinning, following a factorial design which was used mainly to study the effect of four process parameters (PLA solution concentration, PAni solution concentration, applied voltage, and flow rate) on fiber diameter. Due to the non‐Gaussian spread of the fiber diameters, probability density functions (PDF's) were used to describe them, and the main effect analysis was performed considering the most representative values of these PDF's. This study suggests that the main factor which governs the fiber dimension is the concentration of the PAni solution and its synergy with the others factors. Furthermore, in spite of the high complexity of the electrospinning process, the statistical model was able to describe the process with 95% confidence, therefore enabling one to precisely predict fiber diameter. Nanofiber formation was simulated using the results of the experimental design and validation tests were performed to minimize the fiber diameter.

     

Enhanced Directed Evolution in Mammalian Cells Yields a Hyperefficient Pyrrolysyl tRNA for Noncanonical Amino Acid Mutagenesis

Heterologous tRNAs used for noncanonical amino acid (ncAA) mutagenesis in mammalian cells typically show poor activity. We recently introduced a virus-assisted directed evolution strategy (VADER) that can enrich improved tRNA mutants from naïve libraries in mammalian cells. However, VADER was limited to processing only a few thousand mutants; the inability to screen a larger sequence space precluded the identification of highly active variants with distal synergistic mutations. Here, we report VADER2.0, which can process significantly larger mutant libraries. It also employs a novel library design, which maintains base-pairing between distant residues in the stem regions, allowing us to pack a higher density of functional mutants within a fixed sequence space. VADER2.0 enabled simultaneous engineering of the entire acceptor stem of M. mazei pyrrolysyl tRNA (tRNA^Pyl), leading to a remarkably improved variant, which facilitates more efficient incorporation of a wider range of ncAAs, and enables facile development of viral vectors and stable cell-lines for ncAA mutagenesis.