Phosphorylation of Ago2 is required for its role in DNA double-strand break repair.

Repair of DNA double-strand break (DSB) is critical for the maintenance of genome integrity. A class of DSB-induced small RNAs (diRNAs) has been shown to play an important role in DSB repair. In humans, diRNAs are associated with Ago2 and guide the recruitment of Rad51 to DSB sites to facilitate repair by homologous recombination (HR). Ago2 activity has been reported to be regulated by phosphorylation under normal and hypoxic conditions. However, the role of Ago2 phosphorylation in DNA damage repair is unexplored. Here, we show that S672, S828, T830, and S831 of human Ago2 are phosphorylated in response to ionizing radiation (IR). S672A mutation of Ago2 leads to significant reduction in Rad51 foci formation and HR efficiency. We further show that defective association of Ago2 S672A variant with DSB sites, instead of defects in diRNA and Rad51 binding, may account for decreased Rad51 foci formation and HR efficiency. Our study reveals a novel regulatory mechanism for the function of Ago2 in DNA repair.

Protective Effects of Amino Acids on Plasmid DNA Damage Induced by Therapeutic Carbon Ions.

Radioprotectors with few side effects are useful for carbon-ion therapy, which directly induces clustering damage in DNA. With the aim of finding the most effective radioprotector, we investigated the effects of selected amino acids which might have chemical DNA-repair functions against therapeutic carbon ions. In the current study, we employed five amino acids: tryptophan (Trp), cysteine (Cys), methionine (Met), valine (Val) and alanine (Ala). Samples of supercoiled pBR322 plasmid DNA with a 17 mM amino acid were prepared in TE buffer (10 mM Tris, 1 mM ethylenediaminetetraacetic acid, pH 7.5). Phosphate buffered saline (PBS) was also used in assays of the 0.17 mM amino acid. The samples were irradiated with carbon-ion beams (290 MeV/u) on 6 cm spread-out Bragg peak at the National Institute of Radiological Sciences and Heavy Ion Medical Accelerator in Chiba, Japan. Breaks in the DNA were detected as changes in the plasmids and quantified by subsequent electrophoresis on agarose gels. DNA damage yields and protection factors for each amino acid were calculated as ratios relative to reagent-free controls. Trp and Cys showed radioprotective effects against plasmid DNA damage induced by carbon-ion beam, both in PBS and TE buffer, comparable to those of Met. The double-strand break (DSB) yields and protective effects of Trp were comparable to those of Cys. The yields of both single-strand breaks and DSBs correlated with the scavenging capacity of hydroxyl radicals (rate constant for scavenging hydroxyl radicals multiplied by the amino acid concentration) in bulk solution. These data indicate that the radioprotective effects of amino acids against plasmid DNA damage induced by carbon ions could be explained primarily by the scavenging capacity of hydroxyl radicals. These findings suggest that some amino acids, such as Trp, Cys and Met, have good potential as radioprotectors for preventing DNA damage in normal tissues in carbon-ion therapy.

A photoenhanced oxidation of amino acids and the cross-linking of lysozyme mediated by tetrazolium salts.

Tetrazolium salts (TZs) are pervasively utilized as precursors in the dye industry, colorimetric probes in enzyme assays and for exploring nanomaterial toxicity, but its own toxicity is not investigated enough so far. Using femtosecond transient absorption spectroscopy, nanosecond pulse radiolysis (ns-PRL), western blotting and UV-vis absorption spectroscopy, here we characterized a neutral tetrazolinyl radical (with the same maximum absorption at 420 nm and different lifetimes of 5.0 and 9.0 µs for two selected TZs), the key intermediate of TZs reduction, and noticed TZs-formazan production under UV light irradiation accompanied by 41% increase in the cross-linking of lysozyme (Lyso, model protein) compared to TZs-free sample, which uncovered the photoenhanced oxidation of TZs towards Lyso. The ns-PRL in a reductive atmosphere simulated the electron/proton donors of amino acid residues in Lyso upon photoexcitation and revealed the reduction mechanism of TZs, as that first followed one-electron-transfer and then probably proton-coupled electron transfer. This is the first time to report on the photoenhanced oxidation mechanism of TZs, which would provide new insights into the applications of TZs in cell biology, "click" chemistry and nanotoxicology.

Photoinduced cross-linking of formulation buffer amino acids to monoclonal antibodies.

The correct choice of formulation buffer is a critical aspect of drug development and is chosen primarily to improve the stability of a protein therapeutic and protect against degradation. Amino acids are frequently incorporated into formulation buffers. In this study we have identified and characterized light induced cross-links between the side chain of histidine residues in an IgG4 monoclonal antibody and different amino acids commonly used in formulation buffers. These reactions have the potential to impact the overall product quality of the drug. The structure of each cross-link identified was elucidated using high performance liquid chromatography (HPLC) hyphenated to tandem mass spectrometry (MS/MS) with higher energy collisional dissociation (HCD). Furthermore, we speculate on the role of amino acids in formulation buffers and their influence on mAb stability. We theorize that whilst the adduction of formulation buffer amino acids could have a negative impact on product quality, it may protect against other pathways of photo-degradation.

Microwave-Induced Ugi-Four Component Reactions: Synthesis of New Hetero- Steroid-Amino Acid Conjugates.

BACKGROUND: Aza-steroids are an important class of compounds because of their numerous biological activities. The hetero steroids have different hydrogen bonding ability and hydrophobicity in comparison to steroids. MATERIALS AND METHODS: Microwave-induced synthesis of a novel type of hybrid hetero-steroid amine conjugates, following Ugi-four component reactions of steroidal amines with alanine and valine methyl esters as amino acid residues is described. Specifically, hetero-steroid-amino acid conjugate based on D-ring fused hetero steroidal amine, hetero-steroid-amino acid conjugate based on A-ring hetero steroidal amine, and hetero-steroidamino acid conjugate based on B-ring hetero steroidal amine are synthesized. RESULTS AND DISCUSSION: The yield of the products under microwave-induced process was considerably higher than that obtained by the conventional method. In contrast to the conventional method for the synthesis of these molecules, microwave-induced method has several advantages. CONCLUSION: These include rapid reaction, a superior yield of the product, minimum side reaction, and economical microwave-induced process.

Computer and Experimental Simulation of Alloxazine Synthesis from Gamma Irradiation of Amino Acids on Iceland Spar: A Prebiotic Chemistry Perspective.

On ancient Earth, environmental conditions favored prebiotic chemical reactions. In the Archean, some molecules with conjugated rings might have been synthesized, displaying structural stability in the Archean in the presence of ionizing radiation and hydration-dehydration events. Additionally, it is suggested that on ancient Earth, calcite was a common mineral promoting organic compound synthesis. In the present work a study of the interaction of amino acid mixtures with the (104) surface of calcite is presented. Our preliminary results show the abiotic synthesis of alloxazine (a flavin with relevant photochemical properties). Computer simulations were performed in HyperChem 8.0.1. by means of MM+ molecular mechanics and PM3 semi-empirical methods, in 27 possible amino acid trimers of alanine, glycine and lysine. Alloxazine formation is possible by the gamma irradiation of amino acids. The computer simulations show that trimers GGG and GGA promote the further transformation from diketopiperazines (DKP's) and KGK to alloxazine. The computer simulations with free radicals are not stable when alloxazine is interacting with the calcite surface. Experiments in anoxygenic environments with hydration-dehydration events in gamma irradiated samples allow the abiotic formation of flavins, DKP's and a heterocycle compound with possible relevance in prebiotic chemistry.

Seasonal Variation of Mycosporine-Like Amino Acids in Three Subantarctic Red Seaweeds.

UV-absorbing compounds, such as mycosporine-like amino acids (MAAs), are a group of secondary metabolites present in many marine species, including red seaweeds. In these organisms, the content and proportion of the composition of MAAs vary, depending on the species and several environmental factors. Its high cosmetic interest calls for research on the content and composition of MAAs, as well as the dynamics of MAAs accumulation in seaweeds from different latitudes. Therefore, this study aimed to survey the content of UV-absorbing MAAs in three Subantarctic red seaweeds during a seasonal cycle. Using spectrophotometric and HPLC techniques, the content and composition of MAAs of intertidal Iridaea tuberculosa, Nothogenia fastigiate, and Corallina officinalis were assessed. Some samples were also analyzed using high-resolution mass spectrometry coupled with HPLC-ESI-MS in order to identify more precisely the MAA composition. I. tuberculosa exhibited the highest MAA values (above 1 mg g-1 of dried mass weight), while C. officinalis showed values not exceeding 0.4 mg g-1. Porphyra-334 was the main component in N. fastigiata, whereas I. tuberculosa and C. officinalis exhibited a high content of palythine. Both content and composition of MAAs varied seasonally, with high concentration recorded in different seasons, depending on the species, i.e., winter (I. tuberculosa), spring (N. fastigiata), and summer (C. officinalis). HPLC-ESI-MS allowed us to identify seven different MAAs. Two were recorded for the first time in seaweeds from Subantarctic areas (mycosporine-glutamic acid and palythine-serine), and we also recorded an eighth UV-absorbing compound which remains unidentified.

The primary photo-dissociation dynamics of amino acids in aqueous solution: breaking the Cα-bond.

We report a study of the primary photo-dissociation dynamics of aqueous alanine, isoleucine and proline by 200 nm UV pump-IR probe transient absorption spectroscopy. Photo-dissociation of the three amino acids predominantly results in decarboxylation, and 38 ± 7% of the excited alanine, 35 ± 10% of the excited isoleucine and 47 ± 10% of the excited proline zwitterions remain dissociated 100 picoseconds after the excitation. The decarboxylation occurs from a transient intermediate with a lifetime of ∼20 picoseconds to which we assign the excited state of the amino acids based on comparison of the measured and calculated IR spectra, and calculated excited state energy surfaces.

Femtosecond bond breaking and charge dynamics in ultracharged amino acids.

Historically, structure determination of nanocrystals, proteins, and macromolecules required the growth of high-quality crystals sufficiently large to diffract X-rays efficiently while withstanding radiation damage. The development of the X-ray free-electron laser has opened the path toward high resolution single particle imaging, and the extreme intensity of the X-rays ensures that enough diffraction statistics are collected before the sample is destroyed by radiation damage. Still, recovery of the structure is a challenge, in part due to the partial fragmentation of the sample during the diffraction event. In this study, we use first-principles based methods to study the impact of radiation induced ionization of six amino acids on the reconstruction process. In particular, we study the fragmentation and charge rearrangement to elucidate the time scales involved and the characteristic fragments occurring.

A tryptophan synchronous and normal fluorescence study on bacteria inactivation mechanism.

The UV photodissociation kinetics of tryptophan amino acid, Trp, attached to the membrane of bacteria, Escherichia coli and Bacillus subtilis, have been studied by means of normal and synchronous fluorescence. Our experimental data suggest that the fluorescence intensity of Trp increases during the first minute of irradiation with 250 nm to ∼ 280 nm, 7 mW/cm2 UV light, and subsequently decreases with continuous irradiation. During this short, less than a minute, period of time, 70% of the 107 cell per milliliter bacteria are inactivated. This increase in fluorescence intensity is not observed when tryptophan is in the free state, namely, not attached to a protein, but dissolved in water or saline solution. This increase in fluorescence is attributed to the additional fluorescence of tryptophan molecules formed by protein unfolding, the breakage of the bond that attaches Trp to the bacterial protein membrane, or possibly caused by the irradiation of 2 types of tryptophan residues that photolyze with different quantum yields.

Ultrasensitive Quantification of Recombinant Proteins Using AAA-MS.

Recombinant proteins are essential components of therapeutic, biotechnological, food, and household products. In some cases, recombinant proteins must be purified and their quantity and/or concentration precisely determined. In this chapter, we describe a protocol for the quantification of purified recombinant proteins. The protocol is based on a microwave-assisted acidic hydrolysis of the target protein followed by high-resolution mass spectrometry (HRMS) analysis of the hydrolytic products. Absolute quantification is obtained by adding controlled amounts of labeled amino acids that serve as standards.

Reversible photo-cross-linking of the GCN4 peptide containing 3-cyanovinylcarbazole amino acid to double-stranded DNA.

Interaction analysis in vivo greatly promotes the analyses and understanding of biological functions. The interaction between DNA and peptides or proteins is very important in terms of readout and amplifying information from genomic DNA. In this study, we designed and synthesized a photo-cross-linkable amino acid, l-3-cyanovinlycarbazole amino acid (l-CNVA), to double-stranded DNA. Reversible photo-cross-linking between DNA and peptides containing CNVA, having 3-cyanovinylcarbazole moieties capable of photo-cross-linking to nucleic acids, was demonstrated. As a result, it was shown that the GCN4 peptide, containing CNVA, can be photo-cross-linked to DNA, and its adduct was photo-split into the original peptide and DNA with 312 nm-irradiation. This is the first report that reversibly manipulates photo-crosslinking between double stranded DNA and peptides. In addition, this reversible photo-cross-linking, using l-CNVA, is faster and with higher yield than that using diazirine and psoralen.

Global gene expression changes reflecting pleiotropic effects of Irpex lacteus induced by low--intensity electromagnetic field.

A polysaccharide of Irpex lacteus, a white-rot fungus with lignocellulose-degrading activities, has been used as a commercial medicine for nephritis treatment. Previously, a low-intensity electromagnetic field (LI-EMF) was found to increase the biomass and polysaccharide content of Irpex lacteus and induce twists on the cell surface. In this study, RNA-sequencing (RNA-seq) technology was used to analyze the underlying mechanism of LI-EMF's influence on Irpex lacteus. We identified 3268, 1377, and 941 differentially expressed genes (DEGs) in the LI-EMF-treated samples at recovery times of 0 h, 3 h, and 6 h, respectively, indicating a significant decline in the influence of the LI-EMF treatment on Irpex lacteus with the passage of recovery time. Moreover, 30 upregulated and 14 downregulated DEGs overlapped in the LI-EMF-treated samples at the recovery times of 0 h, 3 h, and 6 h, implying the important lasting effects of LI-EMF. The reliability of the RNA-seq data were validated by quantitative real-time PCR (qRT-PCR). The DEGs related to transcription factors, cell proliferation, cell wall, membrane components, amino acid biosynthesis and metabolism, and polysaccharide biosynthesis and metabolism were significantly enriched in the LI-EMF-treated samples. The experiments confirmed that the LI-EMF treatment significantly increased the content of amino acids with a considerable increase in the content of essential amino acids. Therefore, the global gene expression changes explained the pleiotropic effects of Irpex lacteus induced by the LI-EMF treatment. These findings provide the requisite data for the appropriate design and application of LI-EMF in the fermentation of microorganisms to increase production. Bioelectromagnetics. 40:104-117, 2019. © 2019 Bioelectromagnetics Society.

The chronic effect of pulsed 1800 MHz electromagnetic radiation on amino acid neurotransmitters in three different areas of juvenile and young adult rat brain.

The extensive use of mobile phones worldwide has raised increasing concerns about the effects of electromagnetic radiation (EMR) on the brain due to the proximity of the mobile phone to the head and the appearance of several adverse neurological effects after mobile phone use. It has been hypothesized that the EMR-induced neurological effects may be mediated by amino acid neurotransmitters. Thus, the present study investigated the effect of EMR (frequency 1800 MHz, specific absorption rate 0.843 W/kg, power density 0.02 mW/cm2, modulated at 217 Hz) on the concentrations of amino acid neurotransmitters (glutamic acid, aspartic acid, gamma aminobutyric acid, glycine, taurine, and the amide glutamine) in the hippocampus, striatum, and hypothalamus of juvenile and young adult rats. The juvenile and young adult animals were each divided into two groups: control rats and rats exposed to EMR 1 h daily for 1, 2, and 4 months. A subgroup of rats were exposed daily to EMR for 4 months and then left without exposure for 1 month to study the recovery from EMR exposure. Amino acid neurotransmitters were measured in the hippocampus, striatum, and hypothalamus using high-performance liquid chromatography. Exposure to EMR induced significant changes in amino acid neurotransmitters in the studied brain areas of juvenile and young adult rats, being more prominent in juvenile animals. It could be concluded that the alterations in amino acid neurotransmitters induced by EMR exposure of juvenile and young adult rats may underlie many of the neurological effects reported after EMR exposure including cognitive and memory impairment and sleep disorders. Some of these effects may persist for some time after stopping exposure.

Employing Photoredox Catalysis for DNA-Encoded Chemistry: Decarboxylative Alkylation of α-Amino Acids.

A new procedure for the photoredox-mediated conjugate addition of radicals that can be conveniently generated from α-amino acids to DNA-tagged Michael acceptors and styrenes is presented. This C(sp3 )-C(sp3 ) coupling tolerates a broad array of structurally diverse radical precursors, including all of the 20 proteinogenic amino acids. Importantly, this reaction proceeds under mild conditions and in DNA-compatible aqueous media. Furthermore, the presented reaction conditions are compatible with DNA, making this reaction platform well suited for the construction of DNA-encoded libraries. The scope and limitations of the chemistry are discussed herein along with proposals for how this methodology might be used to construct DNA-encoded libraries.

Probing Ion Channel Structure and Function Using Light-Sensitive Amino Acids.

Approaches to remotely control and monitor ion channel operation with light are expanding rapidly in the biophysics and neuroscience fields. A recent development directly introduces light sensitivity into proteins by utilizing photosensitive unnatural amino acids (UAAs) incorporated using the genetic code expansion technique. The introduction of UAAs results in unique molecular level control and, when combined with the maximal spatiotemporal resolution and poor invasiveness of light, enables direct manipulation and interrogation of ion channel functionality. Here, we review the diverse applications of light-sensitive UAAs in two superfamilies of ion channels (voltage- and ligand-gated ion channels; VGICs and LGICs) and summarize existing UAA tools, their mode of action, potential, caveats, and technical considerations to their use in illuminating ion channel structure and function.

Dissociation of biomolecules in liquid environments during fast heavy-ion irradiation.

The effect of aqueous environment on fast heavy-ion radiation damage of biomolecules was studied by comparative experiments using liquid- and gas-phase amino acid targets. Three types of amino acids with different chemical structures were used: glycine, proline, and hydroxyproline. Ion-induced reaction products were analyzed by time-of-flight secondary-ion mass spectrometry. The results showed that fragments from the amino acids resulting from the C-Cα bond cleavage were the major products for both types of targets. For liquid-phase targets, specific products originating from chemical reactions in solutions were observed. Interestingly, multiple dissociated atomic fragments were negligible for the liquid-phase targets. We found that the ratio of multifragment to total fragment ion yields was approximately half of that for gas-phase targets. This finding agreed with the results of other studies on biomolecular cluster targets. It is concluded that the suppression of molecular multifragmentation is caused by the energy dispersion to numerous water molecules surrounding the biomolecular solutes.

Photorelaxation and Photorepair Processes in Nucleic and Amino Acid Derivatives.

Understanding the fundamental interaction between electromagnetic radiation and matter is essential for a large number of phenomena, with significance to civilization.[...].

Effect of Pulsed Light Irradiation on Bioactive, Nonvolatile Components and Antioxidant Properties of Caterpillar Medicinal Mushroom Cordyceps militaris (Ascomycetes).

The caterpillar medicinal mushroom Cordyceps militaris contains many bioactive components, such as adenosine, cordycepin, and polysaccharides. In this study, C. militaris was exposed to 0, 3, 6, or 9 pulses of light irradiation to estimate changes in vitamin D2, bioactive compounds, nonvolatile taste components, and antioxidant properties. In addition, we compared the components and properties of C. militaris mycelia and solid waste medium that had been treated with pulsed light (PL) irradiation. Overall, PL irradiation of C. militaris increased the vitamin D2 content and increased the total amino acid levels 9-48%; the antioxidant properties of the mycelia treated with 0 pulses and of the solid waste medium treated with 3 pulses all exhibited lower half-maximal effective concentrations. Therefore, PL irradiation affected the amounts of bioactive compounds, but the irradiated samples still contained intense umami taste and a sufficient amount of antioxidant components.