Ratiometric fluorescence nanoscopy and lifetime imaging of novel Nile Red analogs for analysis of membrane packing in living cells.

Subcellular membranes have complex lipid and protein compositions, which give rise to organelle-specific membrane packing, fluidity, and permeability. Due to its exquisite solvent sensitivity, the lipophilic fluorescence dye Nile Red has been used extensively to study membrane packing and polarity. Further improvement of Nile Red can be achieved by introducing electron-donating or withdrawing functional groups. Here, we compare the potential of derivatives of Nile Red with such functional substitutions for super-resolution fluorescence microscopy of lipid packing in model membranes and living cells. All studied Nile Red derivatives exhibit cholesterol-dependent fluorescence changes in model membranes, as shown by spectrally resolved stimulated emission depletion (STED) microscopy. STED imaging of Nile Red probes in cells reveals lower membrane packing in fibroblasts from healthy subjects compared to those from patients suffering from Niemann Pick type C1 (NPC1) disease, a lysosomal storage disorder with accumulation of cholesterol and sphingolipids in late endosomes and lysosomes. We also find small but consistent changes in the fluorescence lifetime of the Nile Red derivatives in NPC1 cells, suggesting altered hydrogen-bonding capacity in their membranes. All Nile Red derivatives are essentially non-fluorescent in water but increase their brightness in membranes, allowing for their use in MINFLUX single molecule tracking experiments. Our study uncovers the potential of Nile Red probes with functional substitutions for nanoscopic membrane imaging.

Whole blood multiplex measurements using electrochemical aptamer-based biosensors.

Simultaneous measurements of various molecules ("multiplex") using electrochemical biosensors typically require multiple electrode implementations, which for neonates, hemophiliacs, etc. is problematic. Here, we introduce the oxazine ATTO 700 into electrochemical aptamer-based biosensors to achieve "true" multiplex, continuous and real-time measurements of two different molecules in undiluted whole blood using a single electrode.

Simultaneous quantification of five antiretrovirals in human tissues using ultra-high performance liquid chromatography-tandem mass spectrometry methods for therapeutic drug monitoring at the sites of action.

Although antiretroviral therapy (ART) is highly effective for the treatment of HIV-1 infection to suppress virus in the blood, HIV persists in tissues. HIV persistence in the tissues is due to numerous factors, and one of those factors are antiretroviral (ARV) concentrations. ARV concentrations in tissues must be adequate to suppress HIV at the sites of action. While therapeutic drug monitoring in the plasma is well-known, drug monitoring in the tissues provides local assessments of adequate ARV exposure to prevent localized HIV resistance formation. Towards these efforts, we validated an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) method in human tissues (cervical, rectal, and vaginal tissues) for the simultaneous quantification of five ARVs: bictegravir, cabotegravir, dolutegravir, doravirine, and raltegravir. For this assay, protein precipitation with acetonitrile with stable, isotopically-labeled internal standards followed by supernatant pre-concentration was performed. Analyte separation was accomplished using a multistep UPLC gradient mixture of 0.1 % formic acid in water (A) and acetonitrile (B) with a Waters Cortecs T3 (2.1x100 mm) column. The assay was extensively validated as per the United States Food and Drug Administration Bioanalytical Method Validation Guidance over a clinically observed range (0.05-50 ng/mL) with superb linearity (R2 > 0.99 across all ARVs). The assay run time was 8.5 min. This analytical method achieves appropriate performance of trueness (85.5-107.4 %), repeatability, and precision (CV < 15 %). Our method will be employed for the therapeutic monitoring of guideline-recommended ARVs in human tissues for monitoring therapeutic efficacy in HIV treatment and prevention research efforts.

Recognition of 8-Oxo-2'-deoxyguanosine in DNA Using the Triphosphate of 2'-Deoxycytidine Connecting the 1,3-Diazaphenoxazine Unit, dCdapTP.

DNA is constantly damaged by various external and internal factors. In particular, oxidative damage occurs in a steady state, and 8-oxo-2'-deoxyguanosine (oxodG) is known as the main oxidative damage. OxodG is a strong genotoxic nucleoside and is thought to be involved in the pathogenesis of cancer and neurological diseases. However, a breakthrough method to detect the position of oxodG in DNA has not yet been developed. Therefore, we attempted to develop a novel method to detect oxodG in DNA using artificial nucleosides. Recently, we have succeeded in the recognition of oxodG in DNA by a single nucleotide elongation reaction using nucleoside derivatives based on a purine skeleton with a 1,3-diazaphenoxazine unit. In this study, we developed a new nucleoside derivative with a pyrimidine skeleton in order to further improve the recognition ability and enzymatic reaction efficiency. We, therefore, designed and synthesized 2'-deoxycytidine-1,3-diazaphenoxazine (Cdap) and its triphosphate derivatives. The results showed that it was incorporated into the primer strand relative to the dG template because of its cytidine skeleton, but it was more effective at the complementary position of the oxodG template. These results indicate that the new nucleoside derivative can be considered as one of the new candidates for the detection of oxodG in DNA.

A targeted fluorescent nanosensor for ratiometric pH sensing at the cell surface.

The correlation between altered extracellular pH and various pathological conditions, including cancer, inflammation and metabolic disorders, is well known. Bulk pH measurements cannot report the extracellular pH value at the cell surface. However, there is a limited number of suitable tools for measuring the extracellular pH of cells with high spatial resolution, and none of them are commonly used in laboratories around the world. In this study, a versatile ratiometric nanosensor for the measurement of extracellular pH was developed. The nanosensor consists of biocompatible polystyrene nanoparticles loaded with the pH-inert reference dye Nile red and is surface functionalized with a pH-responsive fluorescein dye. Equipped with a targeting moiety, the nanosensor can adhere to cell membranes, allowing direct measurement of extracellular pH at the cell surface. The nanosensor exhibits a sensitive ratiometric pH response within the range of 5.5-9.0, with a calculated pKa of 7.47. This range optimally covers the extracellular pH (pHe) of most healthy cells and cells in which the pHe is abnormal, such as cancer cells. In combination with the nanosensors ability to target cell membranes, its high robustness, reversibility and its biocompatibility, the pHe nanosensor proves to be well suited for in-situ measurement of extracellular pH, even over extended time periods. This pH nanosensor has the potential to advance biomedical research by improving our understanding of cellular microenvironments, where extracellular pH plays an important role.

Alared: Solvatochromic and Fluorogenic Red Amino Acid for Ratiometric Live-Cell Imaging of Bioactive Peptides.

To fill the need for environmentally sensitive fluorescent unnatural amino acids able to operate in the red region of the spectrum, we have designed and synthesized Alared, a red solvatochromic and fluorogenic amino acid derived from the Nile Red chromophore. The new unnatural amino acid can be easily integrated into bioactive peptides using classical solid-phase peptide synthesis. The fluorescence quantum yield and the emission maximum of Alared-labeled peptides vary in a broad range depending on the peptide's environment, making Alared a powerful reporter of biomolecular interactions. Due to its red-shifted absorption and emission spectra, Alared-labeled peptides could be followed in living cells with minimal interference from cellular autofluorescence. Using ratiometric fluorescence microscopy, we were able to track the fate of the Alared-labeled peptide agonists of the apelin G protein-coupled receptor upon receptor activation and internalization. Due to its color-shifting environmentally sensitive emission, Alared allowed for distinguishing the fractions of peptides that are specifically bound to the receptor or unspecifically bound to different cellular membranes.

Colorimetric and fluorometric determination of uric acid by a suspension-based assay using enzyme-immobilized micro-sized particles.

Daily monitoring of serum uric acid levels is very important to provide appropriate treatment according to the constitution and lifestyle of individual hyperuricemic patients. We have developed a suspension-based assay to measure uric acid by adding a sample solution to the suspension containing micro-sized particles immobilized on uricase and horseradish peroxidase (HRP). In the proposed method, the mediator reaction of uricase, HRP, and uric acid produces resorufin from Amplex red. This resorufin is adsorbed onto enzyme-immobilized micro-sized particles simultaneously with its production, resulting in the red color of the micro-sized particles. The concentration of resorufin on the small surface area of the microscopic particles achieves a colorimetric analysis of uric acid with superior visibility. In addition, ethanol-induced desorption of resorufin allowed quantitative measurement of uric acid using a 96-well fluorescent microplate reader. The limit of detection (3σ) and RSD (n = 3) were estimated to be 2.2 × 10-2 µg/mL and ≤ 12.1%, respectively. This approach could also be applied to a portable fluorometer.

Construction of a novel near-infrared fluorescent Nile blue@MOF nanoprobe for imaging mitochondrial ATP in living cells.

A near-infrared fluorescent nanoprobe consisting of Nile blue-capped ZIF-90 is first proposed for real-time imaging of mitochondrial ATP. Owing to the strong binding of ATP with Zn2+, the structure of the probe is disrupted, leading to the release of fluorescent NB.

Discovery of Dolutegravir Derivative against Liver Cancer via Inducing Autophagy and DNA Damage.

We introduced a terminal alkyne into the core structure of dolutegravir, resulting in the synthesis of 34 novel dolutegravir-1,2,3-triazole compounds through click chemistry. These compounds exhibited remarkable inhibitory activities against two hepatocellular carcinoma cell lines, Huh7 and HepG2. Notably, compounds 5e and 5p demonstrated exceptional efficacy, particularly against Huh7 cells, with IC50 values of 2.64 and 5.42 µM. Additionally, both compounds induced apoptosis in Huh7 cells, suppressed tumor cell clone formation, and elevated reactive oxygen species (ROS) levels, further promoting tumor cell apoptosis. Furthermore, compounds 5e and 5p activated the LC3 signaling pathway, inducing autophagy, and triggered the γ-H2AX signaling pathway, resulting in DNA damage in tumor cells. Compound 5e exhibited low toxicity, highlighting its potential as a promising anti-tumor drug.

Site-specific incorporation of a fluorescent nucleobase analog enhances i-motif stability and allows monitoring of i-motif folding inside cells.

The i-motif is an intriguing non-canonical DNA structure, whose role in the cell is still controversial. Development of methods to study i-motif formation under physiological conditions in living cells is necessary to study its potential biological functions. The cytosine analog 1,3-diaza-2-oxophenoxazine (tCO) is a fluorescent nucleobase able to form either hemiprotonated base pairs with cytosine residues, or neutral base pairs with guanines. We show here that when tCO is incorporated in the proximity of a G:C:G:C minor groove tetrad, it induces a strong thermal and pH stabilization, resulting in i-motifs with Tm of 39ºC at neutral pH. The structural determination by NMR methods reveals that the enhanced stability is due to a large stacking interaction between the guanines of the tetrad with the tCO nucleobase, which forms a tCO:C+ in the folded structure at unusually-high pHs, leading to an increased quenching in its fluorescence at neutral conditions. This quenching is much lower when tCO is base-paired to guanines and totally disappears when the oligonucleotide is unfolded. By taking profit of this property, we have been able to monitor i-motif folding in cells.

Natural products with 1,2-oxazine scaffold: occurrence, chemical diversity, bioactivity, synthesis, and biosynthesis.

Covering: up to the end of July, 20231,2-Oxazine is a heterocyclic scaffold rarely found in natural products and is characterized by a directly connected N-O bond in a six-membered ring. Since the discovery of geneserine, the first 1,2-oxazine-containing natural product (1,2-oxazine NP) being isolated from Calabar bean (Physostigma venenosum) in 1925, a total of 76 naturally occurring 1,2-oxazine NPs have been isolated and identified from various sources, which have attracted the attention of researchers in the field of natural product chemistry, organic synthesis, biosynthesis, and pharmacology. This review summarizes the chemical family of 1,2-oxazine NPs, focusing on their source organisms, structural diversities, chemical synthesis, and biosynthesis.

Metal-free anomalous [5+1] cycloaddition reactions of donor-acceptor aziridines for the synthesis of 2H-1,4-oxazines.

A new type of metal-free [5+1] cycloaddition reaction of donor-acceptor aziridines with 2-(2-isocyanoethyl)indoles is reported herein. This method exhibits broad substrate scope and atom-economy. A series of 2H-1,4-oxazines containing an indole heterocycle skeleton were obtained in up to 92% yield under mild reaction conditions. Control experiments revealed that free indole N-H is crucial for the above transformations. The theoretical calculation studies provided guidance on the in-depth insight into the reaction mechanism and the hydrogen-bond between the free indole N-H and carbonyl group was identified to lower the free energy barrier in the transition states.

Antifouling Properties of Poly(2-Oxazoline)s and Poly(2-Oxazine)s: Direct Comparison of Polymer-Coated Surfaces with the Same Coating Parameters.

This study presents a systematic comparison of the antifouling properties of water-soluble poly(2-oxazoline) (PAOx) and poly(2-oxazine) (PAOzi) brushes grafted to gold surfaces. PAOx and PAOzi are emerging polymer classes in biomedical sciences and are being considered superior alternatives to widely used polyethylene glycol (PEG). Four different polymers, poly(2-methyl-2-oxazoline) (PMeOx), poly(2-ethyl-2-oxazoline) (PEtOx), poly(2-methyl-2-oxazine) (PMeOzi), and poly(2-ethyl-2-oxazine) (PEtOzi), each of them in three different chain lengths, are synthesized and characterized for their antifouling properties. Results show that all polymer-modified surfaces display better antifouling properties than bare gold surfaces as well as analogous PEG coatings. The antifouling properties increase in the following order: PEtOx < PMeOx ≈ PMeOzi < PEtOzi. The study suggests that the resistance to protein fouling derives from both surface hydrophilicity and the molecular structural flexibility of the polymer brushes. PEtOzi brushes with moderate hydrophilicity show the best antifouling performance, possibly due to their highest chain flexibility. Overall, the research contributes to the understanding of antifouling properties in PAOx and PAOzi polymers, with potential applications in various biomaterials.

Pretrichodermamide A Biosynthesis Reveals the Hidden Diversity of Epidithiodiketopiperazines.

Fungal epidithiodiketopiperazines (ETPs) possess large structural diversity and complexity due to modifications of the cyclodipeptide skeleton. Elucidation of the biosynthetic pathway of pretrichodermamide A (1) in Trichoderma hypoxylon revealed a flexible catalytic machinery of multiple enzymes for generating ETP diversity. Seven tailoring enzymes encoded by the tda cluster are involved in 1 biosynthesis, that is, four P450s TdaB and TdaQ for 1,2-oxazine formation, TdaI for C7'-hydroxylation, and TdaG for C4, C5-epoxidation, two methyltransferases TdaH for C6'- and TdaO for C7'-O-methylation, and a reductase TdaD for furan opening. Gene deletions led to the identification of 25 novel ETPs, including 20 shunt products, indicating the catalytic promiscuity of Tda enzymes. Particularly, TdaG and TdaD accept various substrates and catalyze regiospecific reactions at different stages of 1 biosynthesis. Our study not only uncovers a hidden library of ETP alkaloids, but also helps to understand the hidden chemical diversity of natural products by pathway manipulation.

Resazurin Reduction-Based Assays Revisited: Guidelines for Accurate Reporting of Relative Differences on Metabolic Status.

Cell viability and metabolic activity are ubiquitous parameters used in biochemistry, molecular biology, and biotechnological studies. Virtually all toxicology and pharmacological projects include at some point the evaluation of cell viability and/or metabolic activity. Among the methods used to address cell metabolic activity, resazurin reduction is probably the most common. At variance with resazurin, resorufin is intrinsically fluorescent, which simplifies its detection. Resazurin conversion to resorufin in the presence of cells is used as a reporter of metabolic activity of cells and can be detected by a simple fluorometric assay. UV-Vis absorbance is an alternative technique but is not as sensitive. In contrast to its wide empirical "black box" use, the chemical and cell biology fundamentals of the resazurin assay are underexplored. Resorufin is further converted to other species, which jeopardizes the linearity of the assays, and the interference of extracellular processes has to be accounted for when quantitative bioassays are aimed at. In this work, we revisit the fundamentals of metabolic activity assays based on the reduction of resazurin. Deviation to linearity both in calibration and kinetics, as well as the existence of competing reactions for resazurin and resorufin and their impact on the outcome of the assay, are addressed. In brief, fluorometric ratio assays using low resazurin concentrations obtained from data collected at short time intervals are proposed to ensure reliable conclusions.

Differential effects of thiamethoxam and clothianidin exposure on their tissue distribution and chronic toxicity in mice.

The frequent application of second-generation neonicotinoids thiamethoxam (TMX) and clothianidin (CLO) has led to a high detectable rate in environment samples and poses threats to nontarget organisms and human beings, however, the information on the influences of long-term exposure at low doses was limited. In this study, the tissue distribution of TMX and CLO in mice at acceptable daily intake (ADI) level and 5 × ADI was determined and the health effects were assessed. TMX and CLO were detected in the liver, serum, lung, heart and kidney in the TMX exposure groups, which indicated that TMX degraded to CLO in mice. Residue levels of TMX in tissues increased with the increasing of doses. The concentrations of CLO in different tissues in the CLO exposure groups were in the order Ckidney > Clung > Cheart > Cliver. Measurement of biochemical indicators, combined with metabolomic analysis of liver, kidney, and cecal contents, examination of changes in the gut microbiota, and histopathological assessment indicated that both TMX and CLO affected energy absorption and lipid metabolism in mice and destroyed tissue structures. Furthermore, we found that CLO had a stronger effect on metabolism in mice, despite its lower acute toxicity. These results have prompted us to consider the chronic toxicity and potential hazards of chemicals in future risk assessments.

Spirooxindol-1,3-oxazine Alkaloids: Highly Potent and Selective Antitumor Agents Evolved from Iterative Structure Optimization.

Spirooxindole-1,3-oxazines are a small and structurally unique class of spirooxindole alkaloids. To date, only four of these compounds have been isolated from natural sources, and their biological properties remained unknown thus far. Dioxyreserpine is a synthetic spirooxindole-1,3-oxazine, that can readily be prepared from the Rauvolfia alkaloid (-)-reserpine by catalytic photooxygenation. While dioxyreserpine itself was now identified as a moderately effective antitumoral agent, structurally modified analogs of it emerged as a new class of highly potent and selective growth inhibitors of various human cancers, including pancreatic cancers. Systematic structural optimization ultimately led to an inhibitor displaying low-micromolar IC50 -values against six cancer cell lines as well as selective apoptosis induction in vitro.

Derivatizing Nile Red fluorophores to quantify the heterogeneous polarity upon protein aggregation in the cell.

Protein aggregation in the cell is often manifested by the formation of subcellular punctate structures. Herein, we modulated the solvatochromism and solubility of Nile Red fluorophore derivatives to quantitatively study the polarity inside pathogenic protein aggregates, revealing structure- and protein-dependent polarity heterogeneity.

Design and synthesis of novel nitrothiazolacetamide conjugated to different thioquinazolinone derivatives as anti-urease agents.

The present article describes the design, synthesis, in vitro urease inhibition, and in silico molecular docking studies of a novel series of nitrothiazolacetamide conjugated to different thioquinazolinones. Fourteen nitrothiazolacetamide bearing thioquinazolinones derivatives (8a-n) were synthesized through the reaction of isatoic anhydride with different amine, followed by reaction with carbon disulfide and KOH in ethanol. The intermediates were then converted into final products by treating them with 2-chloro-N-(5-nitrothiazol-2-yl)acetamide in DMF. All derivatives were then characterized through different spectroscopic techniques (1H, 13C-NMR, MS, and FTIR). In vitro screening of these molecules against urease demonstrated the potent urease inhibitory potential of derivatives with IC50 values ranging between 2.22 ± 0.09 and 8.43 ± 0.61 µM when compared with the standard thiourea (IC50 = 22.50 ± 0.44 µM). Compound 8h as the most potent derivative exhibited an uncompetitive inhibition pattern against urease in the kinetic study. The high anti-ureolytic activity of 8h was confirmed against two urease-positive microorganisms. According to molecular docking study, 8h exhibited several hydrophobic interactions with Lys10, Leu11, Met44, Ala47, Ala85, Phe87, and Pro88 residues plus two hydrogen bound interactions with Thr86. According to the in silico assessment, the ADME-Toxicity and drug-likeness profile of synthesized compounds were in the acceptable range.

An Optical Sensing Platform for Beta-Glucosidase Activity Using Protein-Inorganic Hybrid Nanoflowers.

In this work, a convenient and dual-signal readout optical sensing platform for the sensitively and selectively determination of beta-glucosidase (ß-Glu) activity was reported using protein-inorganic hybrid nanoflowers [BSA-Cu3(PO4)2·3H2O] possessing peroxidase-mimicking activity. The nanoflowers (NFs) were facilely synthesized through a self-assembled synthesis strategy at room temperature. The as-prepared NFs could catalytically convert the colorless and non-fluorescent Amplex Red into colored and highly fluorescent resorufin in the presence of hydrogen peroxide via electron transfer process. ß-Glu could hydrolyze cyanogenic glycoside, using amygdalin (Amy) as a model, into cyanide ions (CN-), which can subsequently efficiently suppress the catalytic activity of NFs, accompanied with the fluorescence decrease and the color fading. The concentration of CN- was controlled by ß-Glu-triggered enzymatic reaction of Amy. Thus, a sensing system was established for fluorescent and visual determination of ß-Glu activity. Under the optimum conditions, the present fluorescent and visual bimodal sensing platform exhibited good sensitivity for ß-Glu activity assay with a detection limit of 0.33 U·L-1. The sensing platform was further applied to determinate ß-Glu in real samples and satisfactory results were attained. Additionally, the optical sensing system can potentially be a promising candidate for ß-Glu inhibitors screening.