Crosslinking of base modified RNAs by synthetic DYW-KP base editors implicates an enzymatic lysine as the nitrogen donor for U-to-C RNA editing.

Base editing mechanisms are being investigated as potential therapeutic tools to alleviate genetic diseases. Sequence specific C-to-U and A-to-I base editing tools are capable of altering RNA and DNA sequences and utilize a hydrolytic deamination mechanism requiring an active site zinc ion and a glutamate residue. In plant organelles, DYW-PG domain containing enzymes catalyze C-to-U edits and likely use the canonical deamination mechanism. Proteins developed from consensus sequences from the DYW-KP domain family catalyze what initially appeared to be U-to-C edits leading to this investigation into mechanistic insights into U-to-C editing. The synthetic DYW-KP enzyme KP6 was found sufficient for C-to-U editing activity stimulated by the addition of carboxylic acids in vitro. Despite addition of 14 different putative amine/amide donors in vitro U-to-C editing could not be observed. C-to-U editing was found not to be concomitant with U-to-C editing thus discounting a pyrimidine transaminase mechanism. RNAs containing base modifications were covalently crosslinked to KP6, KP2, and KP3 proteins. Mass spectrometry of purified KP2 and KP6 proteins revealed an additional mass of 319 Da. A U-to-C crosslinking mechanism was projected to explain the link between crosslinking, RNA base changes, and the ∼319 Da mass. In this model an enzymatic lysine attacks C4 of uridine to form a Schiff base RNA-protein conjugate. Sequenced RT-PCR products from the fern Ceratopteris richardii indicate U-to-C base edits do not preserve proteinaceous crosslinks in planta. Hydrolysis of a protonated Schiff base conjugate releasing cytidine is hypothesized to explain the completed pathway in plants.

Chemistry of Singlet Oxygen with a Cadmium-Sulfur Cluster: Physical Quenching versus Photooxidation.

We investigated the chemistry of singlet oxygen with a cadmium-sulfur cluster, (Me4N)2[Cd4(SPh)10]. This cluster was used as a model for cadmium-sulfur nanoparticles. Such nanoparticles are often used in conjunction with photosensitizers (for singlet oxygen generation or dye-sensitized solar cells), and hence, it is important to determine if cadmium-sulfur moieties physically quench and/or chemically react with singlet oxygen. We found that (Me4N)2[Cd4(SPh)10] is indeed a very strong quencher of singlet oxygen with total rate constants for 1O2 removal of (5.8 ± 1.3) × 108 M-1 s-1 in acetonitrile and (1.2 ± 0.5) × 108 M-1 s-1 in CD3OD. Physical quenching predominates, but chemical reaction leading to decomposition of the cluster and formation of sulfinate is also significant, with a rate constant of (4.1 ± 0.6) × 106 M-1 s-1 in methanol. Commercially available cadmium-sulfur quantum dots ("lumidots") show similar singlet oxygen quenching rate constants, based on the molar concentration of the quantum dots.

Nano in nano: Biosynthesized gold and iron nanoclusters cargo neoplastic exosomes for cancer status biomarking.

Timely detection is crucial for successful treatment of cancer. The current study describes a new approach that involves utilization of the tumor cell environment for bioimaging with in-situ biosynthesized nanoscale gold and iron probes and subsequent dissemination of Au-Fe nanoclusters from cargo exosomes within the circulatory system. We have isolated the Au-Fe cargo exosomes from the blood of the treated murine models after in situ biosyntheses from their respective pre-ionic solutions (HAuCl4, FeCl2), whereas Na2SeO3 supplementation added into Au lethal effect. The microarray data of various differentially expressed genes revealed the up-regulated tumor ablation and metal binding genes in SGC-7901 cell lines after treatment with Au-Fe-Se triplet ionic solution. The isolation of Au-Fe nanoclusters cargo exosomes (nano in nano) after secretion from deeply seated tumors may help in early diagnosis and reveal the tumor ablation status during and after the relevant treatment like radio-chemo therapies et al.

Biosynthesized Gold Nanoclusters and Iron Complexes as Scaffolds for Multimodal Cancer Bioimaging.

Cancer treatment has a far greater chance of success if the neoplasm is diagnosed before the onset of metastasis to vital organs. Hence, cancer early diagnosis is extremely important and remains a major challenge in modern therapeutics. In this contribution, facile and new method for rapid multimodal tumor bioimaging is reported by using biosynthesized iron complexes and gold nanoclusters via simple introduction of AuCl4- and Fe2+ ions. The observations demonstrate that the biosynthesized Au nanoclusters may act as fluorescent and computed tomography probes for cancer bioimaging while the iron complexes behave as effective contrast agent for magnetic resonance imaging. The biosynthesized iron complexes and gold nanoclusters are found biocompatible in vitro (MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay) and in vivo for all the vital organs of circulatory and excretory system. These observations raise the possibility that the biosynthesized probes may find applications in future clinical diagnosis for deep seated early neoplasms by multimodal imaging.

A mechanistic paradigm for broad-spectrum antivirals that target virus-cell fusion.

LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC50 ≤ 0.5 µM), and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001's specific inhibition of virus-cell fusion. The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen ((1)O2) quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated (1)O2. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. (1)O2-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced (1)O2-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity) critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides. Based on our understanding of LJ001's mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001's limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR) studies led to a novel class of compounds (oxazolidine-2,4-dithiones) with (1) 100-fold improved in vitro potency (IC50<10 nM), (2) red-shifted absorption spectra (for better tissue penetration), (3) increased quantum yield (efficiency of (1)O2 generation), and (4) 10-100-fold improved bioavailability. Candidate compounds in our new series moderately but significantly (p≤0.01) delayed the time to death in a murine lethal challenge model of Rift Valley Fever Virus (RVFV). The viral membrane may be a viable target for broad-spectrum antivirals that target virus-cell fusion.

Sequential photooxidation of a Pt(II) (diimine)cysteamine complex: intermolecular oxygen atom transfer versus sulfinate formation.

The thiolato complex [platinum(II) (bipyridine)(N,S-aminoethanethiolate)](+)Ch(-) (1) undergoes sequential reactions with singlet oxygen to initially form the corresponding sulfenato complex [platinum(II) (bipyridine)(N,S(═O)-aminoethansulfenate)](+) (2) followed by a much slower reaction to the corresponding sulfinato complex. In contrast with many platinum dithiolato complexes, 1 does not produce any singlet oxygen, but its rate constant for singlet oxygen removal (k(T)) is quite large (3.2 × 10(7) M(-1) s(-1)) and chemical reaction accounts for ca. 25% of the value of k(T). The behavior of 1 is strikingly different from that of the complex platinum(II) (bipyridine)(1,2-benzenditholate) (4). The latter complex reacts with (1)O(2) (either from an external sensitizer or via a self-sensitized pathway) to form a sulfinato complex. These two very different reactivity pathways imply different mechanistic pathways: The reaction of 1 with (1)O(2) must involve O-O bond cleavage and intermolecular oxygen atom transfer, while the reactive intermediate in complex 4 collapses intramolecularly to the sulfinato moiety.

Singlet Oxygen Quenching by Resveratrol Derivatives.

We investigated the singlet oxygen quenching ability of several derivatives of trans-resveratrol which have been reported to have significant antioxidant ability, including photoprotective activity. We measured the total rate constants of singlet oxygen removal (kT ) by the methylated resveratrol derivative 1,3-dimethoxy-5-[(E)-2-(4-methoxyphenyl)ethenyl]benzene, and the partially methylated resveratrol derivatives 4-((E)-2-(3,5-dimethoxyphenyl)ethenyl)phenol (pterostilbene), 5-[(E)-2-(4-methoxyphenyl)ethenyl]benzene-1,3-diol and (2R,3R)-3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-2,3-dihydrochromen-4-one (dihydromyricetin). A protic solvent system results in higher kT values, except for the completely methylated derivative. We also investigated the ability of trans-resveratrol to directly act as a photosensitizer (rather than via secondary photoproducts resulting from other primary photochemical reactions) for the production of singlet oxygen but found that neither resveratrol nor any of its derivatives are able to do so. We then studied the chemical reactions of the methylated derivative with singlet oxygen. The main pathway consists of a [4 + 2] cycloaddition reaction involving the trans-double bond and the para-substituted benzene ring similar to what has been observed for trans-resveratrol. Unlike trans-resveratrol, the primary singlet oxygen product undergoes a second [4 + 2] cycloaddition with singlet oxygen leading to the formation of diendoperoxides. A second reactivity pathway for both trans-resveratrol and the methylated derivative leads to the formation of aldehydes via cleavage of a transient dioxetane.

New strategy of efficient inhibition of cancer cells by carborane carboxylic acid-CdTe nanocomposites.

Nanoconjugates composed of drug molecules encapsulated in quantum dots (QDs) attract enormous attention due to their promising bioimaging and biomedical applications. Here, the anticancer efficiency of potential pharmacophore agents (o-carborane (Cb), o-carborane-C-carboxylic acid (Cbac1), and o-carborane-C(1)C(2)-dicarboxylic acid (Cbac2) coupling with cadmium telluride QDs capped with cysteamine (CA-CdTe QDs)) have been explored. Compared with free CA-CdTe QDs, the composites consisting of Cbac1/Cbac2 and safe-dosage QDs can greatly improve the inhibition efficiency toward SMMC-7721 hepatocellular carcinoma cells with the aid of our real-time cell bioelectronic sensing system and the MTT assay. The enhanced cytotoxicity correlates with increased intracellular reactive oxygen species generation and cell apoptosis. Confocal laser scanning fluorescent microscopy shows improved cellular uptake and drug distribution of the Cbac1/Cbac2-CdTe QDs nanoconjugates. This work raises the possibility that the carborane pharmacophore in combination with QDs or other anticancer drugs may be viable for efficient cancer diagnosis and chemotherapy.

Bioactivity of the conjugation of green-emitting CdTe quantum dots with a carborane complex.

In this report, we describe the effect of conjugating o-carborane-C(1)C(2)-dicarboxylic acid (o-C2B10H10-C2O4H2, denoted as Cbac2) to cadmium telluride quantum dots (CdTe QDs) capped with cysteamine on the photophysics and cytotoxicity of the QDs. Cbac2 quenches the fluorescence intensity and induces a red shift of the fluorescence emission peak. Meanwhile, studies with a real time cell electronic sensing (RT-CES) system and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay indicate that the combination of the carborane carboxylic acid derivative Cbac2 with relevant QDs can efficiently improve the inhibition efficiency for target cancer cells when compared with a single ligand or the CdTe QDs alone. This study raises the possibility for the labeling of the important pharmacophore with QDs and the design of new promising anticancer agents containing the carborane pharmacophores for cancer therapy.

Singlet Oxygen Generation, Quenching and Reactivity with Metal Thiolates.

Metal thiolate complexes can act as photosensitizers for the generation of singlet oxygen, quenchers of singlet oxygen, and they may undergo chemical reactions with singlet oxygen leading to oxidized thiolate ligands. This review covers all of the chemical reactions of thiolate ligands with singlet oxygen (through early 2021). Since some of these reactions are self-sensitized photooxidations, singlet oxygen generation by metal complexes is also discussed. Mechanistic features such as the effects of protic vs. aprotic conditions are presented and compared with the comparatively well-understood photooxidation of organic sulfides. In general, the total rate of singlet oxygen removal correlates with the nucleophilicity of the thiolate ligand which in turn can be influenced by the metal. Some interesting patterns of reactivity have been noted as a result of this survey: Metal thiolate complexes bearing arylthiolate ligands appear to exclusively produce sulfinate (metal-bound sulfone) products upon reaction with singlet oxygen. In contrast, metal thiolate complexes bearing alkylthiolate ligands may produce sulfinate and/or sulfenate (metal-bound sulfoxide) products. Several mechanistic pathways have been proposed for these reactions, but the exact nature of any intermediates remains unknown at this time.

Probing the dynamic effect of cys-CdTe quantum dots toward cancer cells in vitro.

The application of quantum dots (QDs) in various biomedical areas requires detailed studies of their toxicity. We report a new strategy for probing the biocompatibility of these nanocrystals, namely, a dynamic investigation of cellular uptake images, cell growth curves, metabolic activity changes, and apoptosis aspects of cadmium telluride QDs capped with cysteamine (Cys-CdTe QDs) on human hepatocellular carcinoma SMMC-7721 cells. We used a real-time cell electronic sensing (RT-CES) system in combination with fluorescence microscopy, 3-(4,5-dimethyl-thiazol-zyl)-2,5-diphenyltetrazolium bromide assay, and flow cytometry (FCM) analysis. As observed from fluorescence images and RT-CES system results, Cys-CdTe QDs can readily bind on the cell plasma membrane and then enter into the cancer cell, causing decreased adherence of cancer cells during the initial 6-12 h, while the metabolic activity apparently decreased. After 24 h, the metabolic activity of the cancer cells was significantly reduced, with continued reduction in metabolic activity observed at even longer incubation times. Moreover, FCM observation and DNA fragmentation analysis clearly indicate apoptosis-related phenomena when SMMC-7721 cells were treated with the Cys-CdTe QDs. Thus, our study reveals details of the cellular aging and death process induced by Cys-CdTe QDs.

A Photoprotective Effect by Cation-π-Interaction? Quenching of Singlet Oxygen by an Indole Cation-π Model System.

We investigated the effect of the cation-π interaction on the susceptibility of a tryptophan model system toward interaction with singlet oxygen, that is, type II photooxidation. The model system consists of two indole units linked to a lariat crown ether to measure the total rate of removal of singlet oxygen by the indole units in the presence of sodium cations (i.e. indole units subject to a cation-π interaction) and in the absence of this interaction. We found that the cation-π interaction significantly decreases the total rate of removal of singlet oxygen (kT ) for the model system, that is, (kT  = 2.4 ± 0.2) × 108  m-1  s-1 without sodium cation vs (kT  = 6.9 ± 0.9) × 107  m-1  s-1 upon complexation of sodium cation to the crown ether. Furthermore, we found that the indole moieties undergo type I photooxidation processes with triplet excited methylene blue; this effect is also inhibited by the cation-π interaction. The chemical rate of reaction of the indole groups with singlet oxygen is also slower upon complexation of sodium cation in our model system, although we were unable to obtain an exact ratio due to differences of the chemical reaction rates of the two indole moieties.

An integrated microfluidic device for large-scale in situ click chemistry screening.

An integrated microfluidic device has been developed to perform 1024 in situ click chemistry reactions in parallel using the bovine carbonic anhydrous II (bCAII) click chemistry system as a proof-of-concept study and a rapid hit identification approach using SPE purification and electrospray-ionization mass spectrometry, multiple reaction monitoring (MRM) analysis, all of which improves the sensitivity and throughput of the downstream analysis.

A dynamic micromixer for arbitrary control of disguised chemical selectivity.

A new type of dynamic micromixer combining the concepts of parallel multi-lamination and hydrodynamic focusing was developed for arbitrary control of disguised chemical selectivity.

Photooxidation of metal-bound thiolates: reactivity of sulfur containing peroxidic intermediates.

A variety of reactions of singlet oxygen with metal-bound thiolates are described, and contrasted with the photooxidation of organic sulfides. Superficially, these two processes appear to involve similar mechanisms, but there are important differences: unlike the photooxidation of organic sulfides, the rate of the initial reaction of metal-thiolates with singlet oxygen (k(t)) appears to be affected by protic solvents and acids. The nucleophilicity of the thiolate moiety is reduced by addition of acids or in protic solvents, leading to significantly lower k(t) values. The primary intermediate in the photooxidation of organic sulfides is a nucleophilic persulfoxide, which can be stabilized by protic solvents or by addition of acid. However, the primary intermediate in the photooxidation of metal thiolates cannot be trapped with phosphite, suggesting that it may be less nucleophilic than its organic counterpart. Support for this hypothesis is also derived from the rather modest (compared with organic sulfides) acceleration of the rate of product formation by addition of acid.

Mammalian cells: a unique scaffold for in situ biosynthesis of metallic nanomaterials and biomedical applications.

Production of nanoscale materials often requires the use of toxic chemicals and complex synthetic procedures. A new scaffold has been explored for in situ synthesis of nanomaterials that utilizes natural biological systems in the form of plants, bacteria, fungi, algae and redox-imbalanced mammalian cells and systems. The latter approach has become popular in recent years and has shown some promising results in bioimaging of cancer, as well as inflammatory and neurodegenerative maladies. Biosynthesis of nanoclusters in redox-imbalanced mammalian cells is facile, cost-effective and environmentally friendly with higher biocompatibility and target specificity and lower adverse effects than traditional synthetic approaches. Herein, we describe recent advances in mammalian green in situ biosynthesis for biomedical applications, especially in cancer and neurodegenerative disease theranostics.

Intramolecular arene epoxidation by phosphadioxiranes.

Singlet oxygen reacts with binaphthyl phosphine derivatives such as 1,1'-binaphthyl di-tert-butyl phosphine to form the corresponding binaphthyl-2-oxide phosphine oxides. This new intramolecular arene epoxidation reaction proceeds with complete retention of stereochemistry. The binaphthyl-2-oxide di-tert-butyl phosphine oxide undergoes a slow "NIH-rearrangement" to form the corresponding hydroxylated product. A transient phosphadioxirane intermediate has been directly observed by low-temperature NMR. Kinetic analyses show that all of the phosphadioxirane intermediate is converted to product. [reaction: see text]

Photooxidation of Co-thiolato complexes in protic and aprotic solvents.

Protic solvents decrease the susceptibility of the thiolate ligand in Co(III) thiolato complexes toward attack by singlet oxygen, but greatly increase the conversion of the peroxidic intermediate to the sulfenato product.

Protective effect of TiO2 nanowhiskers on Tetra Sulphonatophenyl Porphyrin (TSPP) complexes induced oxidative stress during photodynamic therapy.

BACKGROUND: Tetra Sulphonatophenyl Porphyrin (TSPP) is well known photosensitizer for photodynamic therapy; nevertheless, its well-known adverse effects hamper its potential use. Recently, nano TiO2's potential role in biomedical has been defined for various disease theranostics, including cancer and other infections. Thus, in this contribution we have explored the possibility of utilizing TiO2 nanowhiskers as novel strategy to lower TSPP adverse effects both in vitro, and in vivo. METHODS: Various concentrations of TSPP, TiO2-TSPP, and TiO2 were injected to three different rat groups, while fourth group was kept as control. Toxic effects were evaluated on excretory and circulatory system by using histopathology, fluorescent microscopy, complete blood cells count (CBC) and serum enzymes. RESULTS: In complete blood cells count, all cells were significantly (p<0.01) affected by the various concentration and treatment groups. The various dose concentrations and treatment also significantly (p<0.01) affected the serum enzyme parameters including AST, ALT, LDH, Creatinine and BUN level. The low concentration of TSPP-TiO2 was found to be the safest, on the bases of serum enzyme parameters, CBC, histopathology, and fluorescent microscopic analysis. The MTT assay was used to evaluate in vitro cytotoxicity, and the results demonstrated maximum viability in illuminated TSPP-TiO2 nanowhiskers group when compared with TSPP treated group. CONCLUSIONS: It was evident that increase in concentration of TSPP increased the toxic effects; however, the TiO2 nanowhiskers combination with TSPP decreased these adverse effects. Moreover, TSPP (0.1 mM) combined with TiO2 nanowhiskers (0.6 mM) was safer than TSPP (0.1 mM) alone.

In Situ Biosynthesis of Fluorescent Platinum Nanoclusters: Toward Self-Bioimaging-Guided Cancer Theranostics.

Among the noble-metal clusters, very few reports about platinum clusters were used as bioimaging probes of tumors except as a reducing catalyst. It is first established herein that the biocompatible platinum nanoclusters are spontaneously biosynthesized by cancerous cells (i.e., HepG2 (human hepatocarcinoma), A549 (lung cancer), and others) rather than noncancerous cells (i.e., L02 (human embryo liver cells)) when incubated with micromolar chloroplatinic acid solutions. These in situ biosynthesized platinum nanoclusters could be readily realized in a biological environment and emit a bright fluorescence at 460 nm, which could be further utilized to facilitate an excellent cancer-cell-killing efficiency when combined with porphyrin derivatives for photothermal treatment. This raises the possibility of providing a promising and precise bioimaging strategy for specific fluorescent self-biomarking of tumor locations and realizing fluorescence imaging-guided photothermal therapy of tumors.