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The degradation of synthetic dye wastewater is important for green chemistry and cost-effectiveness. In this study, we developed Fe 3 O 4 @C-laccase (laccase immobilized on Fe 3 O 4 @C nanoparticles) for photothermal degradation of high concentration of triphenylmethane dye wastewater. The Fe 3 O 4 @C-laccase possessed superior pH and thermal stabilities, as well as excellent tolerance to organic solvents, inhibitors, and metal ions. Laccase activity assays revealed that the activity recovery was approximately 118.2 %. Furthermore, the Fe 3 O 4 @C-laccase presented rapid and sustainable photothermal degradation capabilities to triphenylmethane dye wastewater. The initial removal efficiencies of 400 mg/L malachite green (MG), 400 mg/L brilliant green (BG), 100 mg/L crystal violet (CV), and 600 mg/L mixed dye (MG:BG:CV = 1:1:1) wastewater were approximately 99.8 %, 99.9 %, 96.4 % and 99.2 % by 60 min treatment, respectively. After undergoing 10 batches of reuse, the photothermal degradation efficiencies of the triphenylmethane dye wastewater remained consistently high, at about 99.3 %, 97.4 %, 94.0 %, and 95.1 %, respectively. The excellent photothermal degradation properties indicate that the Fe 3 O 4 @C-laccase holds promise for addressing high concentration of textile wastewater in various applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Understanding how the structure of molecules affects their permeability across cell membranes is crucial for many topics in biomedical research, including the development of drugs. In this work, we examine the transport rates of structurally similar triphenylmethane dyes, malachite green (MG) and brilliant green (BG), across the membranes of living Escherichia coli (E. coli) cells and biomimetic liposomes. Using the time-resolved second harmonic light scattering technique, we found that BG passively diffuses across the E. coli cytoplasmic membrane (CM) 3.8 times faster than MG. In addition, BG exhibits a diffusion rate 3.1 times higher than MG across the membranes of liposomes made from E. coli polar lipid extracts. Measurements on these two molecules, alongside previously studied crystal violet (CV), another triphenylmethane molecule, are compared against the set of propensity rules developed by Lipinski and co-workers for assessing the permeability of hydrophobic ion-like drug molecules through biomembranes. It indicates that BG's increased diffusion rate is due to its higher lipophilicity, with a distribution coefficient 25 times greater than MG. In contrast, CV, despite having similar lipophilicity to MG, shows negligible permeation through the E. coli CM on the observation scale, attributed to its more hydrogen bonding sites and larger polar surface area. Importantly, cell viability tests revealed that BG's antimicrobial efficacy is ∼2.4 times greater than that of MG, which aligns well with its enhanced diffusion into the E. coli cytosol. These findings offer valuable insights for drug design and development, especially for improving the permeability of poorly permeable drug molecules., (© 2024 Author(s). Published under an exclusive license by AIP Publishing.)

Being a low-toxic and hydrophilic representative of TAM, OX063 has shown its suitability for in-vivo and in-cell EPR experiments and design of spin labels. Using 13 C labeling, we investigated the course of oxidative degradation of OX063 into quinone-methide (QM) under the influence of superoxide as well as further thiol-promoted reduction of QM into TAM radical, which formally corresponds to substitution of a carboxyl function by a hydroxyl group. We found these transformations being quantitative in model reactions mimicking specific features of biological media and confirmed the presence of these reactions in the blood and liver homogenate of mice in vitro. The emergence of the trityl with the hydroxyl group can be masked by an initial TAM in EPR spectra and may introduce distortions into EPR-derived oximetry data if they have been obtained for objects under hypoxia. 13 C labeling allows one to detect its presence, considering its different hyperfine splitting constant on 13 C 1 (2.04 mT) as compared to OX063 (2.30 mT). The potential involvement of these reactions should be considered when using TAM in spin-labeling of biopolymers intended for subsequent EPR experiments, as well as in the successful application of TAM in experiments in vivo and in cell., (© 2024 Wiley-VCH GmbH.)

Bioreduction of spin labels and polarizing agents (generally stable radicals) has been an obstacle limiting the in-cell applications of pulsed electron paramagnetic resonance (EPR) spectroscopy and dynamic nuclear polarization (DNP). In this work, we have demonstrated that two semiquinone methide radicals (OXQM⋅ and CTQM⋅) can be easily produced from the trityl-based quinone methides (OXQM and CTQM) via reduction by various reducing agents including biothiols and ascorbate under anaerobic conditions. Both radicals have relatively low pKa's and exhibit EPR single line signals at physiological pH. Moreover, the bioreduction of OXQM in three cell lysates enables quantitative generation of OXQM⋅ which was most likely mediated by flavoenzymes. Importantly, the resulting OXQM⋅ exhibited extremely high stability in the E.coli lysate under anaerobic conditions with 76- and 14.3-fold slower decay kinetics as compared to the trityl OX063 and a gem-diethyl pyrrolidine nitroxide, respectively. Intracellular delivery of OXQM into HeLa cells was also achieved by covalent conjugation with a cell-permeable peptide as evidenced by the stable intracellular EPR signal from the OXQM⋅ moiety. Owing to extremely high resistance of OXQM⋅ towards bioreduction, OXQM and its derivatives show great application potential in in-cell EPR and in-cell DNP studies for various cells which can endure short-term anoxic treatments., (© 2024 Wiley-VCH GmbH.)

Laccase is a copper-containing polyphenol oxidase that can oxidize phenolic and non-phenolic organic substrates. In the past decades, laccases had received considerable attention because of the ability to degrade various organic substances. Based on the codon preference of the Pichia pastoris expression system, this study optimized the gene structure of the laccase gene Lcc1 from Coprius cinerea through synthetic biology methods. A new gene Lcc1I was synthesized and heterologously expressed in P. pastoris. After 3 days of cultivation in a shake flask at 30°C, the transformants produced at a yield of 890 mg L -1 protein. The highest production level of the recombinant laccase was 2760 U L -1 . The molecular mass of the recombinant laccase was estimated at 60 kDa. The enzyme showed highest activity at pH 3.4 and 45°C. It possessed better stability at higher pH and lower temperature condition. Using 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulphonate (ABTS) as the substrate, the K m and V max values were 0.136 mM and 9778 μM min -1  mg -1 , respectively. The recombinant laccase could directly oxidize some triphenylmethane dyes like leuco-crystal violet (LCV) and leuco-malachite green (LMG). With the help of ABTS mediator, it could oxidize and degrade 77.7% crystal violet (CV) and 79.2% malachite green (MG) within 1 h. Our results indicate that optimization of the laccase gene achieves good expression results in the host system. The dye degradation model constructed in this study may also be applied to the degradation of other organic pollutants and toxic substances, providing new solutions for environmental remediation against the increasingly severe environmental pollution., (© 2024 John Wiley & Sons Ltd.)

The purpose of this study is to evaluate the decolorization ability and detoxification effect of LAC-4 laccase on various types of single and mixed dyes, and lay a good foundation for better application of laccase in the efficient treatment of dye pollutants. The reaction system of the LAC-4 decolorizing single dyes (azo, anthraquinone, triphenylmethane, and indigo dyes, 17 dyes in total) were established. To explore the decolorization effect of the dye mixture by LAC-4, two dyes of the same type or different types were mixed at the same concentration (100 mg/L) in the reaction system containing 0.5 U laccase, and time-course decolorization were performed on the dye mixture. The combined dye mixtures consisted of azo + azo, azo + anthraquinone, azo + indigo, azo + triphenylmethane, indigo + triphenylmethane, and triphenylmethane + triphenylmethane. The results obtained in this study were as follows. Under optimal conditions of 30 °C and pH 5.0, LAC-4 (0.5 U) can efficiently decolorize four different types of dyes. The 24-hour decolorization efficiencies of LAC-4 for 800 mg/L Orange G and Acid Orange 7 (azo), Remazol Brilliant Blue R (anthraquinone), Bromophenol Blue and Methyl Green (triphenylmethane), and Indigo Carmine (indigo) were 75.94%, 93.30%, 96.56%, 99.94%, 96.37%, and 37.23%, respectively. LAC-4 could also efficiently decolorize mixed dyes with different structures. LAC-4 can achieve a decolorization efficiency of over 80% for various dye mixtures such as Orange G + Indigo Carmine (100 mg/L+100 mg/L), Reactive Orange 16 + Methyl Green (100 mg/L+100 mg/L), and Remazol Brilliant Blue R + Methyl Green (100 mg/L+100 mg/L). During the decolorization process of the mixed dyes by laccase, four different interaction relationships were observed between the dyes. Decolorization efficiencies and rates of the dyes that were difficult to be degraded by laccase could be greatly improved when mixed with other dyes. Degradable dyes could greatly enhance the ability of LAC-4 to decolorize extremely difficult-to-degrade dyes. It was also found that the decolorization efficiencies of the two dyes significantly increased after mixing. The possible mechanisms underlying the different interaction relationships were further discussed. Free, but not immobilized, LAC-4 showed a strong continuous batch decolorization ability for single dyes, two-dye mixtures, and four-dye mixtures with different structures. LAC-4 exhibited high stability, sustainable degradability, and good reusability in the continuous batch decolorization. The LAC-4-catalyzed decolorization markedly reduced or fully abolished the toxic effects of single dyes (azo, anthraquinone, and indigo dye) and mix dyes (nine dye mixtures containing four structural types of dyes) on plants. Our findings indicated that LAC-4 laccase had significant potential for use in bioremediation due to its efficient degradation and detoxification of single and mixed dyes with different structural types., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Detection of amyloid β (Aβ) oligomers, regarded as the most toxic aggregated forms of Aβ, can contribute to the diagnosis and treatment of Alzheimer's disease (AD). Thus, the development of imaging probes for in vivo visualization of Aβ oligomers is crucial. However, the structural uncertainty regarding Aβ oligomers makes it difficult to design imaging probes with high sensitivity to Aβ oligomers against highly aggregated Aβ fibrils. In this study, we developed Aβ oligomer-selective fluorescent probes based on triphenylmethane dyes through screening of commercially available compounds followed by structure-activity relationship (SAR) studies on cyclic or acyclic 4-dialkylamino groups. We synthesized 11 triarylmethane-based Aβ oligomer probe (TAMAOP) derivatives. In vitro evaluation of fluorescence properties, TAMAOP-9, which had bulky 4-diisobutylamino groups introduced into three benzenes of a twisted triphenylmethane backbone, showed marked fluorescence enhancement in the presence of Aβ oligomers and demonstrated high selectivity for Aβ oligomers against Aβ fibrils. In docking studies using the Aβ trimer model, TAMAOP-9 bound to the hydrophobic surface and interacted with the side chain of Phe 20 . In vitro section staining revealed that TAMAOP-9 could visualize Aβ oligomers in the brains of AD model mice. An in vivo fluorescence imaging study using TAMAOP-9 showed significantly higher fluorescence signals from the brains of AD model mice than those of age-matched wild-type mice, confirmed by ex vivo section observation. These results suggest that TAMAOP-9 is a promising Aβ oligomer-targeting fluorescent probe applicable to in vivo imaging.

The use of formalin to preserve raw food items such as fish, meat, vegetables etc . is very commonly practiced in the present day. Also, formaldehyde (FA), which is the main constituent of formalin solution, is known to cause serious health issues on exposure. Considering the ill effects of formaldehyde, herein we report synthesis of highly sensitive triphenylmethane based formaldehyde (FA) sensors from a single step reaction of inexpensive reagents namely 4-hydroxy benzaldehyde and 2,6-dimethyl phenol. The synthetic method also provides highly pure product in bulk quantity. The analytical activity of the triphenylmethane sensor 1 with a limit of detection (LOD) value of 2.31 × 10 -6 M for FA was significantly enhanced through induced deprotonation and thereafter a LOD value of 1.82 × 10 -8 M could be achieved. To the best of our knowledge, the LOD value of the deprotonated form (sensor 2) for FA was superior to those of all the FA optical sensors reported so far. The mechanism of sensing was demonstrated by 1 H-NMR titration and recording mass spectra before and after addition of FA to a solution of sensor 2. Both sensor 1 and sensor 2 exhibit quenching in emission upon addition of FA. A fluorescence study also demonstrates enhancement in analytical activity of the sensor upon induced deprotonation. Then the sensor was effectively immobilized into a hydrophilic and biocompatible starch-PVA polymer matrix which enabled detection of FA in a 100% aqueous system reversibly. Again, quick and effective sensing of FA in real food samples (stored fish) with the help of a computational application was demonstrated. The sensors have significant practical applicability as they effectively detect FA in real food samples qualitatively and quantitatively.

Hypoxia, acidosis, and elevated inorganic phosphate concentration are characteristics of the tumor microenvironment in solid tumors. There are a number of methods for measuring each parameter individually in vivo, but the only method to date for noninvasive measurement of all three variables simultaneously in vivo is electron paramagnetic spectroscopy paired with a monophosphonated trityl radical, pTAM/HOPE. While HOPE has been successfully used for in vivo studies upon intratissue injection, it cannot be delivered intravenously due to systemic toxicity and albumin binding, which causes significant signal loss. Therefore, we present HOPE71, a monophosphonated trityl radical derived from the very biocompatible trityl probe, Ox071. Here, we describe a straightforward synthesis of HOPE71 starting with Ox071 and report its EPR sensitivities to pO 2 , pH, and [P i ] with X-band and L-band EPR spectroscopy. We also confirm that HOPE71 lacks albumin binding, shows low cytotoxicity, and has systemic tolerance. Finally, we demonstrate its ability to profile the tumor microenvironment in vivo in a mouse model of breast cancer.

Solid-phase synthesis of cyclic, branched or side-chain-modified peptides typically involves introduction of a residue carrying a temporary side-chain protecting group that undergoes selective on-resin removal. In particular, N α -Fmoc-N ε -(4-methyltriphenylmethyl) (Mtt)-protected lysine and its shorter analogues are commercially available and extensively used in this context. Nevertheless, rapid reliable methods for on-resin removal of Mtt groups in the presence of tert-butyloxycarbonyl (Boc) groups are needed. Current commonly used conditions involve low concentrations (1-3%) of trifluoroacetic acid (TFA) in dichloromethane, albeit adjustment to each specific application is required to avoid premature removal of Boc groups or cleavage from the linker. Hence, a head-to-head comparison of several deprotection conditions was performed. The selected acids represent a wide range of acidity from TFA to trifluoroethanol. Also, on-resin removal of the N-(4-methoxytriphenylmethyl) (Mmt) and O-trityl groups (on serine) was investigated under similar conditions. The mildest conditions identified for Mtt deprotection involve successive treatments with 30% hexafluoroisopropanol (HFIP) or 30% perfluoro-tert-butanol [(CF 3 ) 3 COH] in dichloromethane (3 × 5 or 3 × 15 min, respectively), while 30% HFIP, 30% (CF 3 ) 3 COH, or 10% AcOH-20% trifluoroethanol (TFE) in CH 2 Cl 2 (3 × 5 min) as well as 5% trichloroacetic acid in CH 2 Cl 2 (3 × 2 min) enabled Mmt removal. Treatment with 1% TFA with/without 2% triisopropylsilane added (3 × 5 min), but also prolonged treatment with 30% (CF 3 ) 3 COH (5 × 15 min), led to selective deprotection of an O-Trt group on a serine residue. In all cases, the sequences also contained N-Boc or O-tBu protecting groups, which were not affected by 30% HFIP or 30% (CF 3 ) 3 COH even after a prolonged reaction time of 4 h. Finally, the optimized conditions involving HFIP or (CF 3 ) 3 COH proved applicable also for selective deprotection of a longer resin-bound peptide [i.e., Ac-Gly-Leu-Leu-Lys(Mtt)-Arg(Pbf)-Ile-Lys(Boc)-Ser(tBu)-Leu-Leu-RAM-PS] as well as allowed for an almost complete deprotection of a Dab(Mtt) residue., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Alterations in viscosity of biological fluids and tissues play an important role in health and diseases. It has been demonstrated that the electron paramagnetic resonance (EPR) spectrum of a 13 C-labeled trityl spin probe ( 13 C-dFT) is highly sensitive to the local viscosity of its microenvironment. In the present study, we demonstrate that X-band (9.5 GHz) EPR viscometry using 13 C-dFT provides a simple tool to accurately measure the microviscosity of human blood in microliter volumes obtained from healthy volunteers. An application of low-field L-band (1.2 GHz) EPR with a penetration depth of 1-2 cm allowed for microviscosity measurements using 13 C-dFT in the living tissues from isolated organs and in vivo in anesthetized mice. In summary, this study demonstrates that EPR viscometry using a 13 C-dFT probe can be used to noninvasively and rapidly measure the microviscosity of blood and interstitial fluids in living tissues and potentially to evaluate this biophysical marker of microenvironment under various physiological and pathological conditions in preclinical and clinical settings.

To clarify the contribution of charge delocalization in a lipophilic ion to the efficacy of its permeation through a lipid membrane, we compared the behavior of alkyl derivatives of triphenylphosphonium, tricyclohexylphosphonium and trihexylphosphonium both in natural and artificial membranes. Exploring accumulation of the lipophilic cations in response to inside-negative membrane potential generation in mitochondria by using an ion-selective electrode revealed similar mitochondrial uptake of butyltricyclohexylphosphonium (C 4 TCHP) and butyltriphenylphosphonium (C 4 TPP). Fluorescence correlation spectroscopy also demonstrated similar membrane potential-dependent accumulation of fluorescein derivatives of tricyclohexyldecylphosphonium and decyltriphenylphosphonium in mitochondria. The rate constant of lipophilic cation translocation across the bilayer lipid membrane (BLM), measured by the current relaxation method, moderately increased in the following sequence: trihexyltetradecylphosphonium ([P 6,6,6,14 ]) < triphenyltetradecylphosphonium (C 14 TPP) < tricyclohexyldodecylphosphonium (C 12 TCHP). In line with these results, measurements of the BLM stationary conductance indicated that membrane permeability for C 4 TCHP is 2.5 times higher than that for C 4 TPP. Values of the difference in the free energy of ion solvation in water and octane calculated using the density functional theory and the polarizable continuum solvent model were similar for methyltriphenylphosphonium, tricyclohexylmethylphosphonium and trihexylmethylphosphonium. Our results prove that both cyclic and aromatic moieties are not necessary for lipophilic ions to effectively permeate through lipid membranes., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The identification of aroma composition and key odorants contributing to aroma characteristics of white tea is urgently needed, owing to white tea's charming flavors and significant health benefits. In this study, a total of 238 volatile components were identified in the three subtypes of white teas using headspace solid-phase microextraction (HS-SPME) combined with comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC × GC-TOFMS). The multivariate statistical analysis demonstrated that the contents of 103 volatile compounds showed extremely significant differences, of which 44 compounds presented higher contents in Baihaoyinzhen and Baimudan, while the other 59 compounds exhibited higher contents in Shoumei. The sensory evaluation experiment carried out by gas chromatography-olfactometry/mass spectrometry (GC-O/MS) revealed 44 aroma-active compounds, of which 25 compounds were identified, including 9 alcohols, 6 aldehydes, 5 ketones, and 5 other compounds. These odorants mostly presented green, fresh, floral, fruity, or sweet odors. Multivariate analyses of chemical characterization and sensory evaluation results showed that high proportions of alcohols and aldehydes form the basis of green and fresh aroma characteristic of white teas, and phenylethyl alcohol, γ-Nonalactone, trans-β-ionone, trans-linalool oxide (furanoid), α-ionone, and cis-3-hexenyl butyrate were considered as the key odorants accounting for the different aroma characteristics of the three subtypes of white tea. The results will contribute to in-depth understand chemical and sensory markers associated with different subtypes of white tea, and provide a solid foundation for tea aroma quality control and improvement.

Antibody-drug conjugates (ADCs) constitute an emerging class of anticancer agents that deliver potent payloads selectively to tumors while avoiding systemic toxicity associated with conventional chemotherapeutics. Critical to ADC development is a serum-stable linker designed to decompose inside targeted cells thereby releasing the toxic payload. A protease-cleavable linker comprising a valine-citrulline (Val-Cit) motif has been successfully incorporated into three FDA-approved ADCs and is found in numerous preclinical candidates. Herein, we present a high-yielding and facile synthetic strategy for a Val-Cit linker that avoids extensive protecting group manipulation and laborious chromatography associated with previous syntheses and provides yields that are up to 10-fold higher than by standard methods. This method is easily scalable and takes advantage of cost-effective coupling reagents and high loading 2-chlorotrityl chloride (2-CTC) resin. Modularity allows for introduction of various conjugation handles in final stages of the synthesis. Facile access to such analogues serves to expand the repertoire of available enzymatically cleavable linkers for ADC generation. This methodology empowers a robust and facile library generation and future exploration into linker analogues containing unnatural amino acids as a selectivity tuning tool.

The unprecedented Nazarov cyclization of a model divinyl ketone using phosphonium-based Deep Eutectic Solvents as sustainable non-innocent reaction media is described. A two-level full factorial Design of Experiments was conducted for elucidating the effect of the components of the eutectic mixture and optimizing the reaction conditions in terms of temperature, time, and substrate concentration. In the presence of the Deep Eutectic Solvent (DES) triphenylmethylphosphonium bromide/ethylene glycol, it was possible to convert more than 80% of the 2,4-dimethyl-1,5-diphenylpenta-1,4-dien-3-one, with a specific conversion, into the cyclopentenone Nazarov derivative of 62% (16 h, 60 °C). For the reactions conducted in the DES triphenylmethylphosphonium bromide/acetic acid, quantitative conversions were obtained with percentages of the Nazarov product above 95% even at 25 °C. Surface Responding Analysis of the optimized data furnished a useful tool to determine the best operating conditions leading to quantitative conversion of the starting material, with complete suppression of undesired side-reactions, high yields and selectivity. After optimization, it was possible to convert more than 90% of the model substrate into the desired cyclopentenone with cis percentages up to 77%. Experimental validation of the implemented model confirmed the robustness and the suitability of the procedure, leading to possible further extension to this specific combination of experimental designs to other substrates or even to other synthetic processes of industrial interest.

We demonstrate a series of multitrityl radical compounds where accurate spin-counting by pulsed electron paramagnetic resonance (EPR) can be achieved at X-band (9 GHz) frequencies, even for molecules with very short and flexible linkers. Multiquantum filter experiments, well-known from NMR, were used to count the number of coupled electron spins in these compounds. The six pulse double quantum filter sequence used in EPR for distance determinations in biradicals was used. Precise phase settings to separate higher quantum coherences were achieved by an arbitrary waveform generator. The trityl radicals have narrow spectral width so that homogeneous excitation of all spins by the pulses is possible. The transversal relaxation times of higher quantum coherences of trityl radicals are sufficiently long to allow their detection. Our results on model compounds show the potential of this approach to determine oligomeric states in protein complexes in their native environment using functionalized trityl spin labels.

The understanding of biomolecular function is coupled to knowledge about the structure and dynamics of these biomolecules, preferably acquired under native conditions. In this regard, pulsed dipolar EPR spectroscopy (PDS) in conjunction with site-directed spin labeling (SDSL) is an important method in the toolbox of biophysical chemistry. However, the currently available spin labels have diverse deficiencies for in-cell applications, for example, low radical stability or long bioconjugation linkers. In this work, a synthesis strategy is introduced for the derivatization of trityl radicals with a maleimide-functionalized methylene group. The resulting trityl spin label, called SLIM, yields narrow distance distributions, enables highly sensitive distance measurements down to concentrations of 90 nm, and shows high stability against reduction. Using this label, the guanine-nucleotide dissociation inhibitor (GDI) domain of Yersinia outer protein O (YopO) is shown to change its conformation within eukaryotic cells., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The pathogenic yeast Candida auris has received increasing attention due to its ability to cause fatal infections, its resistance toward important fungicides, and its ability to persist on surfaces including medical devices in hospitals. To brace health care systems for this considerable risk, alternative therapeutic approaches such as antifungal peptides are urgently needed. In clinical wound care, a significant focus has been directed toward novel surgical (wound) dressings as first defense lines against C. auris. Inspired by Cerberus the Greek mythological "hound of Hades" that prevents the living from entering and the dead from leaving hell, the preparation of a gatekeeper hybrid hydrogel is reported featuring lectin-mediated high-affinity immobilization of C. auris cells from a collagen gel as a model substratum in combination with a release of an antifungal peptide drug to kill the trapped cells. The vision is an efficient and safe two-layer medical composite hydrogel for the treatment of severe wound infections that typically occur in hospitals. Providing this new armament to the repertoire of possibilities for wound care in critical (intensive care) units may open new routes to shield and defend patients from infections and clinical facilities from spreading and invasion of C. auris and probably other fungal pathogens., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

UV-VIS spectroscopy analysis of six mixtures containing choline chloride or triphenylmethylphosphonium bromide as the hydrogen bond acceptor (HBA) and different hydrogen bond donors (HBDs, nickel sulphate, imidazole, d-glucose, ethylene glycol, and glycerol) allowed to determine the indirect and direct band-gap energies through the Tauc plot method. Band-gap energies were compared to those relative to known choline chloride-containing deep band-gap systems. The measurements reported here confirmed the tendency of alcohols or Lewis acids to increment band-gap energy when employed as HBDs. Indirect band-gap energy of 3.74 eV was obtained in the case of the triphenylmethylphosphonium bromide/ethylene glycol system, which represents the smallest transition energy ever reported to date for such kind of systems.

Double-electron electron resonance (DEER) can be used to track the structural dynamics of proteins in their native environment, the cell. This method provides the distance distribution between two spin labels attached at specific, well-defined positions in a protein. For the method to be viable under in-cell conditions, the spin label and its attachment to the protein should exhibit high chemical stability in the cell. Here we present low-temperature, trityl-trityl DEER distance measurements on two model proteins, PpiB (prolyl cis-trans isomerase from E. coli ) and GB1 (immunoglobulin G-binding protein), doubly labeled with the trityl spin label, CT02MA. Both proteins gave in-cell distance distributions similar to those observed in vitro, with maxima at 4.5-5 nm, and the data were further compared with in-cell Gd(III)-Gd(III) DEER obtained for PpiB labeled with BrPSPy-DO3A-Gd(III) at the same positions. These results highlight the challenges of designing trityl tags suitable for in-cell distance determination at ambient temperatures on live cells.

The unpleasant stale note is a negative factor hindering the consumption of instant ripened Pu-erh tea products. This study focused on investigating volatile chemicals in instant ripened Pu-erh tea that could mask the stale note via sensory evaluation, gas chromatography-mass spectrometry (GC-MS), and gas chromatography-olfactometry (GC-O) analyses. GC-MS and GC-O analyses showed that linalool, linalool oxides, trans -β-ionone, benzeneacetaldehyde, and methoxybenzenes were the major aroma contributors to the simultaneous distillation and extraction (SDE) extract of instant ripened Pu-erh tea. Sensory evaluation showed that the SDE extract had a strong stale note, which was due to methoxybenzenes. By investigating suppressive interaction among flavour components, the stale note from methoxybenzenes was shown to have reciprocal masking interactions with sweet, floral, and green notes. Moreover, the validation experiment showed that the addition of 40 μg/mL of trans -β-ionone in the instant ripened Pu-erh tea completely masked the stale note and improved the overall aromatic acceptance. These results elucidate the volatile chemicals that could mask the stale note of instant ripened Pu-erh tea products, which might help to develop high quality products made from instant ripened Pu-erh tea.

Obstructing conductive pathways of the channel-forming toxins with targeted blockers is a promising drug design approach. Nearly all tested positively charged ligands have been shown to reversibly block the cation-selective channel-forming protective antigen (PA 63 ) component of the binary anthrax toxin. The cationic ligands with more hydrophobic surfaces, particularly those carrying aromatic moieties, inhibited PA 63 more effectively. To understand the physical basis of PA 63 selectivity for a particular ligand, detailed information is required on how the blocker structural elements (e.g., positively charged and aromatic groups) influence the molecular kinetics of the blocker/channel binding reactions. In this study, we address this problem using the high-resolution single-channel planar lipid bilayer technique. Several structurally distinct cationic blockers, namely per-6-S-(3-amino) propyl-β-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-α-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-β-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-γ-cyclodextrin, methyltriphenylphosphonium ion, and G0 polyamidoamine dendrimer are tested for their ability to inhibit the heptameric and octameric PA 63 variants and PA 63 F427A mutant. The F427 residues form a hydrophobic constriction region inside the channel, known as the "ϕ-clamp." We show that the cationic blockers interact with PA 63 through a combination of forces. Analysis of the binding reaction kinetics suggests the involvement of cation-π, Coulomb, and salt-concentration-independent π-π or hydrophobic interactions in the cationic cyclodextrin binding. It is possible that these blockers bind to the ϕ-clamp and are also stabilized by the Coulomb interactions of their terminal amino groups with the water-exposed negatively charged channel residues. In PA 63 F427A, only the suggested Coulomb component of the cyclodextrin interaction remains. Methyltriphenylphosphonium ion and G0 polyamidoamine dendrimer, despite being positively charged, interact primarily with the ϕ-clamp. We also show that seven- and eightfold symmetric cyclodextrins effectively block the heptameric and octameric forms of PA 63 interchangeably, adding flexibility to the earlier formulated blocker/target symmetry match requirement., (Copyright © 2019. Published by Elsevier Inc.)

S -trityl-l-cysteine ( STLC ) is a well-recognized lead compound known for its anticancer activity owing to its potent inhibitory effect on human mitotic kinesin Eg5. STLC contains two free terminal amino and carboxyl groups that play pivotal roles in binding to the Eg5 pocket. On the other hand, such a zwitterion structure complicates the clinical development of STLC because of the solubility issues. Masking either of these radicals reduces or abolishes STLC activity against Eg5. We recently identified and characterized a new class of nicotinamide adenine dinucleotide-dependent deacetylase isoform 2 of sirtuin protein (SIRT2) inhibitors that can be utilized as cytotoxic agents based on an S -trityl-l-histidine scaffold. Herein, we propose new STLC -derived compounds that possess pronounced SIRT2 inhibition effects. These derivatives contain modified amino and carboxyl groups, which conferred STLC with SIRT2 bioactivity, representing an explicit repurposing approach. Compounds STC4 and STC11 exhibited half maximal inhibitory concentration values of 10.8 ± 1.9 and 9.5 ± 1.2 μM, respectively, against SIRT2. Additionally, introduction of the derivatizations in this study addressed the solubility limitations of free STLC , presumably due to interruption of the zwitterion structure. Therefore, we could obtain drug-like STLC derivatives that work by a new mechanism of action. The new derivatives were designed, synthesized, and their structure was confirmed using different spectroscopic approaches. In vitro and cellular bioassays with various cancer cell lines and in silico molecular docking and solubility calculations of the synthesized compounds demonstrated that they warrant attention for further refinement of their bioactivity.

Folate-based small molecule drug conjugates (SMDCs) are currently under development and have shown promising preclinical and clinical results against various cancers and polycystic kidney disease. Two requisites for response to a folate-based SMDC are (i) folate receptor alpha (FRα) protein is expressed in the diseased tissues, and (ii) FRα in those tissues is accessible and functionally competent to bind systemically administered SMDCs. Here we report on the development of a small molecule reporter conjugate (SMRC), called EC2220, which is composed of a folate ligand for FRα binding, a multilysine containing linker that can cross-link to FRα in the presence of formaldehyde fixation, and a small hapten (fluorescein) used for immunohistochemical detection. Data show that EC2220 produces a far greater IHC signal in FRα-positive tissues over that produced with EC17, a folate-fluorescein SMRC that is released from the formaldehyde-denatured FRα protein. Furthermore, the extent of the EC2220 IHC signal was proportional to the level of FRα expression. This EC2220-based assay was qualified both in vitro and in vivo using normal tissue, cancer tissue, and polycystic kidneys. Overall, EC2220 is a sensitive and effective reagent for evaluating functional and accessible receptor expression in vitro and in vivo .

Dynamic nuclear polarization (DNP) improves signal-to-noise in nuclear magnetic resonance (NMR) spectroscopy. Signal-to-noise in NMR can be further improved with cryogenic sample cooling. Whereas MAS DNP is commonly performed between 25 and 110 K, sample temperatures below 6 K lead to further improvements in sensitivity. Here, we demonstrate that solid effect MAS DNP experiments at 6 K, using trityl, yield 3.2× more sensitivity compared to 90 K. Trityl with solid effect DNP at 6 K yields substantially more signal to noise than biradicals and cross effect DNP. We also characterize cross effect DNP with AMUPol and TEMTriPol-1 biradicals for DNP magic angle spinning at temperatures below 6 K and 7 Tesla. DNP enhancements determined from microwave on/off intensities are 253 from AMUPol and 49 from TEMTriPol-1. The higher thermal Boltzmann polarization at 6 K compared to 298 K, combined with these enhancements, should result in 10,000× signal gain for AMUPol and 2000× gain for TEMTriPol-1. However, we show that AMUPol reduces signal in the absence of microwaves by 90% compared to 41% by TEMTriPol-1 at 7 Tesla as the result of depolarization and other detrimental paramagnetic effects. AMUPol still yields the highest signal-to-noise improvement per unit time between the cross effect radicals due to faster polarization buildup (T 1DNP  = 4.3 s and 36 s for AMUPol and TEMTriPol-1, respectively). Overall, AMUPol results in 2.5× better sensitivity compared to TEMTriPol-1 in MAS DNP experiments performed below 6 K at 7 T. Trityl provides 6.0× more sensitivity than TEMTriPol-1 and 1.9× more than AMUPol at 6 K, thus yielding the greatest signal-to-noise per unit time among all three radicals. A DNP enhancement profile of TEMTriPol-1 recorded with a frequency-tunable custom-built gyrotron oscillator operating at 198 GHz is also included. It is determined that at 7 T below 6 K a microwave power level of 0.6 W incident on the sample is sufficient to saturate the cross effect mechanism using TEMTriPol-1, yet increasing the power level up to 5 W results in higher improvements in DNP sensitivity with AMUPol. These results indicate MAS DNP below 6 K will play a prominent role in ultra-sensitive NMR spectroscopy in the future., (Copyright © 2019. Published by Elsevier Inc.)

Pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) in combination with site-directed spin labeling (SDSL) of proteins and oligonucleotides is a powerful tool in structural biology. Instead of using the commonly employed gem -dimethyl-nitroxide labels, triarylmethyl (trityl) spin labels enable such studies at room temperature, within the cells and with single-frequency electron paramagnetic resonance (EPR) experiments. However, it has been repeatedly reported that labeling of proteins with trityl radicals led to low labeling efficiencies, unspecific labeling and label aggregation. Therefore, this work introduces the synthesis and characterization of a maleimide-functionalized trityl spin label and its corresponding labeling protocol for cysteine residues in proteins. The label is highly cysteine-selective, provides high labeling efficiencies and outperforms the previously employed methanethiosulfonate-functionalized trityl label. Finally, the new label is successfully tested in PDS measurements on a set of doubly labeled Yersinia outer protein O (YopO) mutants.

Solubilizing trityl tags (Trt-oligoLys/Arg) proved applicable to metal-free radical-triggered desulfurization and an Ag-mediated thioester method. Additionally, using the solubilizing trityl tag strategy, synthesis of the influenza BM2 proton channel, which previously required organic solvent-aided native chemical ligation (NCL) and desulfurization due to its low solubility, was achieved without using organic solvents.

Oxygen is essential in physiology and pathophysiology. Electron paramagnetic resonance (EPR) oximetry, using oxygen sensitive paramagnetic materials, could be attractive for measuring oxygen in tissues. The aim of the present study was to assess the properties of lipid nanocapsules (LNCs) loaded with the nitroxide tempo-benzoate (TB) or tetrathiatriarylmethyl (TAM) radicals. LNCs loaded with the EPR probes were successfully prepared by the phase inversion process leading to nanocapsules of about 60 nm. LNCs protected the TB radical against reduction in vitro. The calibration of the EPR line width (LW) as a function of the pO 2 showed a two-fold increase in sensitivity with TAM-LNC compared to hydrophilic trityl radical. The TAM-LNCs were evaluated in vivo. Contrarily to unencapsulated TAM, for which a rapid decrease in EPR signal was observed, the half-life of TAM-LNCs administered in muscles or in tumours exceeded an hour. Carbogen-challenges in mice demonstrated that the TAM-LNCs responded well to changes in oxygen environment. However, the apparent pO 2 values acquired were higher than the expected physiological values. These results warrant further investigation in the formulation of stable nano-objects encapsulating EPR oxygen sensitive probes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Molecular oxygen, reactive oxygen species and free radicals derived from oxygen play important roles in a broad spectrum of physiological and pathological processes. The quantitative measurement of molecular oxygen in tissues by electron paramagnetic resonance (EPR) has great potential for understanding and diagnosing a number of diseases, and for developing and guiding therapies. This requires improvements in the free radical probe systems that sense and report molecular oxygen levels in vivo. We report on the encapsulation of existing free radical probes in lipophilic gel implants: an in-situ-oleogel and an emulgel, based only on well-known, safe excipients for the incorporation of lipophilic and hydrophilic radicals, respectively. The EPR signals of encapsulated radicals were not altered compared to dissolved radicals. The high solubility of oxygen in lipophilic solvents enhanced oxygen sensitivity. The gels extended the lifetime of the radicals in tissues from tens of minutes to many days, simplifying studies with extended series of measurements. The encapsulated radicals showed a good in vivo response to changes in oxygen supply and seem to circumvent concerns from toxicity of the radical probes. These gels simplify the development of new oxygen-sensitive free radical probes for EPR oximetry by making their in vivo stability, persistence and toxicity a function of the encapsulating gel and not a set of additional requirements for the free radical probe., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Protein and peptide prenylation is an essential biological process involved in many signal transduction pathways. Hence, it plays a critical role in establishing many major human ailments, including Alzheimer's disease, amyotrophic lateral sclerosis (ALS), malaria, and Ras-related cancers. Yeast mating pheromone a-factor is a small dodecameric peptide that undergoes prenylation and subsequent processing in a manner identical to larger proteins. Due to its small size in addition to its well-characterized behavior in yeast, a-factor is an attractive model system to study the prenylation pathway. Traditionally, chemical synthesis and characterization of a-factor have been challenging, which has limited its use in prenylation studies. In this chapter, a robust method for the synthesis of a-factor is presented along with a description of the characterization of the peptide using MALDI and NMR. Finally, complete assignments of resonances from the isoprenoid moiety and a-factor from COSY, TOCSY, HSQC, and long-range HMBC NMR spectra are presented. This methodology should be useful for the synthesis and characterization of other mature prenylated peptides and proteins., (© 2019 Elsevier Inc. All rights reserved.)

Accurate distances between two trityl paramagnetic tags site-specifically attached to DNA duplexes were measured by pulsed electron-electron double resonance spectroscopy at 180 and 260 GHz microwave frequencies. Up to a threefold increase in the sensitivity of 260 GHz PELDOR measurements was achieved by using shaped broad-band microwave pulses.

The search for suitable, low-molecular weight photoantimicrobials for use in infection control has strong foundations in conventional antiseptic research from the early-mid 20 th Century. Many examples of dyes exist having conventional antimicrobial activity among the azine, acridine and triphenylmethane families which have since also been found to exhibit photosensitising capabilities. The prior employment of these examples in human antisepsis provides a practical basis in terms of low host toxicity, while extant structure-activity relationships for conventional antimicrobial activity can support the development of similar relationships for photoactivated cell killing. The range of chromophores covered allows progress to be made both in topical and deeper, fluid-involved infections.

Topical ophthalmologic treatments have been facing great challenges with main limitations of low drug bioavailability, due to highly integrative defense mechanisms of the eye. This study rationally devised strategies to increase drug bioavailability by increasing ocular surface residence time of drug-loaded nanoliposomes dispersed within thermo-sensitive hydrogels (Pluronic F-127). Alternatively, we utilized sub-conjunctival injections as a depot technique to localize nanoliposomes. Senicapoc was encapsulated and sustainably released from free nanoliposomes and hydrogels formulations in vitro. Residence time increased up to 12-fold (60 min) with 24% hydrogel formulations, as compared to 5 min for free liposomes, which was observed in the eyes of Sprague-Dawley rats using fluorescence measurements. Pharmacokinetic results obtained from flushed tears, also showed that the hydrogels had greater drug retention capabilities to that of topical viscous solutions for up to 60 min. Senicapoc also remained quantifiable within sub-conjunctival tissues for up to 24 h post-injection.

Soluble oligomers of the β-amyloid peptide, Aβ, are associated with the progression of Alzheimer's disease. Although many small molecules bind to these assemblies, the details of how these molecules interact with Aβ oligomers remain unknown. This paper reports that crystal violet, and other C3 symmetric triphenylmethane dyes, bind to C3 symmetric trimers derived from Aβ 17-36 . Binding changes the color of the dyes from purple to blue, and causes them to fluoresce red when irradiated with green light. Job plot and analytical ultracentrifugation experiments reveal that two trimers complex with one dye molecule. Studies with several triphenylmethane dyes reveal that three N, N-dialkylamino substituents are required for complexation. Several mutant trimers, in which Phe 19 , Phe 20 , and Ile 31 were mutated to cyclohexylalanine, valine, and cyclohexylglycine, were prepared to probe the triphenylmethane dye binding site. Size exclusion chromatography, SDS-PAGE, and X-ray crystallographic studies demonstrate that these mutations do not impact the structure or assembly of the triangular trimer. Fluorescence spectroscopy and analytical ultracentrifugation experiments reveal that the dye packs against an aromatic surface formed by the Phe 20 side chains and is clasped by the Ile 31 side chains. Docking and molecular modeling provide a working model of the complex in which the triphenylmethane dye is sandwiched between two triangular trimers. Collectively, these findings demonstrate that the X-ray crystallographic structures of triangular trimers derived from Aβ can be used to guide the discovery of ligands that bind to soluble oligomers derived from Aβ.

Laccase CotA from Bacillus subtilis 168 was successfully displayed on the membrane of Escherichia coli cells using poly-γ-glutamate synthetase A protein (PgsA) from B. subtilis as an anchoring matrix. Further analyses demonstrated that the fusion protein PgsA/CotA efficiently translocates to the cell surface of E. coli with an enzymatic activity of 65 U/10 8 cells. Surface-displayed CotA was shown to possess improved enzymatic properties compared with those of the wild-type CotA, including higher thermal stability (above 90% activity at 70 °C and nearly 40% activity at 90 °C after 5-h incubation) and stronger inhibitor tolerance (approximately 80 and 65% activity when incubated with 200 and 400 mM NaCl, respectively). Furthermore, the whole-cell system was demonstrated to have high enzymatic activity against anthraquinone dye, Acid Blue 62, triphenylmethane dye, Malachite Green, and azo dye, Methyl Orange with the decolorization percentages of 91, 45, and 75%, after 5-h incubation, respectively.

Oxygenation is one of the most important physiological parameters of biological systems. Low oxygen concentration (hypoxia) is associated with various pathophysiological processes in different organs. Hypoxia is of special importance in tumor therapy, causing poor response to treatment. Triaryl methyl (TAM) derivative radicals are commonly used in electron paramagnetic resonance (EPR) as sensors for quantitative spatial tissue oxygen mapping. They are also known as magnetic resonance imaging (MRI) contrast agents and fluorescence imaging compounds. We report the properties of the TAM radical tris(2,3,5,6-tetrachloro-4-carboxy-phenyl)methyl, (PTMTC), a potential multimodal (EPR/fluorescence) marker. PTMTC was spectrally analyzed using EPR and characterized by estimation of its sensitivity to the oxygen in liquid environment suitable for intravenous injection (1 mM PBS, pH = 7.4). Further, fluorescent emission of the radical was measured using the same solvent and its quantum yield was estimated. An in vitro cytotoxicity examination was conducted in two cancer cell lines, HT-29 (colorectal adenocarcinoma) and FaDu (squamous cell carcinoma) and followed by uptake studies. The stability of the radical in different solutions (PBS pH = 7.4, cell media used for HT-29 and FaDu cells culturing and cytotoxicity procedure, full rat blood and blood plasma) was determined. Finally, a primary toxicity test of PTMTC was carried out in mice. Results of spectral studies confirmed the multimodal properties of PTMTC. PTMTC was demonstrated to be not absorbed by cancer cells and did not interfere with luciferin-luciferase based assays. Also in vitro and in vivo tests showed that it was non-toxic and can be freely administrated till doses of 250 mg/kg BW via both i.v. and i.p. injections. This work illustrated that PTMTC is a perfect candidate for multimodal (EPR/fluorescence) contrast agent in preclinical studies.

Small-conductance Ca 2+ -activated K + (SK) channels mediate neuron excitability and are associated with synaptic transmission and plasticity. They also regulate immune responses and the size of blood cells. Activation of SK channels requires calmodulin (CaM), but how CaM binds and opens SK channels has been unclear. Here we report cryo-electron microscopy (cryo-EM) structures of a human SK4-CaM channel complex in closed and activated states at 3.4- and 3.5-angstrom resolution, respectively. Four CaM molecules bind to one channel tetramer. Each lobe of CaM serves a distinct function: The C-lobe binds to the channel constitutively, whereas the N-lobe interacts with the S4-S5 linker in a Ca 2+ -dependent manner. The S4-S5 linker, which contains two distinct helices, undergoes conformational changes upon CaM binding to open the channel pore. These structures reveal the gating mechanism of SK channels and provide a basis for understanding SK channel pharmacology., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Tris(2,3,5,6-tetrathiaaryl)methyl radicals, so-called trityl radicals, are emerging as spin labels for distance measurements in biological systems based on Electron Paramagnetic Resonance (EPR). Here, the synthesis and characterization of rigid model systems carrying either two or three trityl moieties is reported. The monofunctionalized trityl radicals are connected to the molecular bridging scaffold via an esterification reaction employing the Mukaiyama reagent 2-chloro-methylpyridinium iodide. The bis- and tris-trityl compounds exhibit different inter-spin distances, strength of electron-electron exchange and dipolar coupling and can give rise to multi-spin effects. They are to serve as benchmark systems in comparing EPR distance measurement methods., Competing Interests: The authors declare no conflict of interest.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

Detection of triphenylmethane dyes (TDs), especially the widely used malachite green (MG) and crystal violet (CV), plays an important role in safety control of aquatic products. There are two chromatic forms of TDs: oxidized or reduced. Usually, only one form can be detected by reported ELISA antibodies. In this article, molecular shape superimposing and quantum mechanics calculation were employed to elucidate the differences between MG, CV, and their reduced chromatic forms (leucomalachite green, LMG and leucocrystal violet, LCV). A potential hapten was rationally designed and synthesized. Polyclonal antibodies were raised through immunizing New Zealand white rabbits and BALB/C mice. We tested the cross-reactivity ratios between the hapten and TDs. The cross-reactivity ratios were correlated with the difference in surface electrostatic potential. The determination coefficients ( r ²) of the correlations are 0.901 and 0.813 for the rabbit and mouse antibody, respectively. According to this linear model, the significant difference in the atomic charge seemed to make it impossible to find a hapten that can produce antibodies with good cross-reactivities with both reduced and oxidized TDs., Competing Interests: The authors declare no conflict of interest.

Superoxide radical represents one of the most biologically relevant reactive oxygen species involved in numerous physiological and pathophysiological processes. Superoxide measurement through the decay of an electron paramagnetic resonance (EPR) signal of a triarylmethyl (TAM) radical possesses the advantage of a high selectivity and relatively high rate constant of TAM reaction with the superoxide. Hereby we report a straightforward synthesis and characterization of a TAM-TAM biradical showing a high reactivity with superoxide (second-order rate constant, (6.7 ± 0.2) × 10 3 M -1 s -1 ) enabling the measurement of superoxide radical by following the increase of a sharp EPR signal associated with the formation of a TAM-quinone-methide monoradical product.

The industrial dye-contaminated wastewater has been considered as the most complex and hazardous in terms of nature and composition of toxicants that can cause severe biotic risk. Reactive azo, anthroquinone and triphenylmethane dyes are mostly used in dyeing industries; thus, the unfixed hydrolysed molecules of these dyes are commonly found in wastewater. In this regard, bacterial species have been proved to be highly effective to treat wastewater containing reactive dyes and heavy metals. The bio-decolourisation of dye occurs either by adsorption or through degradation in bacterial metabolic pathways under optimised environmental conditions. The bacterial dye decolourisation rates vary with the type of bacteria, reactivity of dye and operational parameters such as temperature, pH, co-substrate, electron donor and dissolved oxygen concentration. The present paper reviews the efficiency of bacterial species (individual and consortia) to decolourise wastewater containing reactive azo, anthroquinone and triphenylmethane dyes either individually or mixed or with metal ions. It has been observed that bacteria Pseudomonas spp. are comparatively more effective to treat reactive dyes and metal-contaminated wastewater. In recent studies, either immobilised cell or isolated enzymes are being used to decolourise dye at a large scale of operations. However, it is required to investigate more potent bacterial species or consortia that could be used to treat wastewater containing mixed reactive dyes and heavy metals like chromium ions.

In this study, a novel generation of deep eutectic solvents (DESs) was used as an acceptor phase in three-phase hollow fiber liquid phase microextraction (HF-LPME) based on two immiscible organic phases. It was compared with other common DESs for extraction and preconcentration of dydrogesterone (DYD) and cyproterone acetate (CPA) from urine and plasma samples. The extracted analytes were analyzed by high performance liquid chromatography with UV-vis detector (HPLC-UV). This phosphonium based DES due to low volatility, low price and multifunctionality introduced itself as worthy next generation of acceptor phase in HF-LPME. The factors affected on extraction efficiency of the analytes were investigated and optimized. The performance of the proposed method was studied in terms of linear ranges (LRs from 1 to 500µgL -1 with R 2 ≥ 0.9946), precision (RSD% ≤ 6.3) and limits of detection (LODs in the range of 0.5-2µgL -1 ). Under the optimized conditions, preconcentration factors in the range of 187-428 were obtained. Finally, the method was applied to the analysis of DYD and CPA in human urine and plasma samples and desirable results were obtained., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Triarylmethyl (trityl, TAM) radicals are a promising class of spin labels for nanometer-scale distance measurements in biomolecules at physiological temperatures. However, to date, existing approaches to site-directed TAM labeling of DNA have been limited to label attachment at the termini of oligonucleotides, thus hindering a majority of demanded applications. Herein, we report a new versatile strategy for TAM attachment at arbitrary sites of nucleic acids. It utilizes an achiral non-nucleoside phosphoramidite monomer for automated solid-phase synthesis of oligonucleotides, which are then postsynthetically functionalized with TAM. We demonstrate a synthesis of a set of oligonucleotide complexes that are TAM-labeled at internal or terminal sites, as well as the possibility of measuring interspin distances up to ∼5-6 nm at 298 K using double quantum coherence electron paramagnetic resonance (EPR). Implementation of the developed approach strongly broadens the scope of nucleic acids and nucleoprotein complexes available for nanoscale structural EPR studies at room temperatures.

Background: The intermediate-conductance Ca2+-activated K+ channel KCa3.1 is widely expressed in cells of the immune system such as T- and B-lymphocytes, mast cells, macrophages and microglia, but also found in dedifferentiated vascular smooth muscle cells, fibroblasts and many cancer cells including pancreatic, prostate, leukemia and glioblastoma. In all these cell types KCa3.1 plays an important role in cellular activation, migration and proliferation by regulating membrane potential and Ca2+ signaling., Methods and Results: KCa3.1 therefore constitutes an attractive therapeutic target for diseases involving excessive proliferation or activation of one more of these cell types and researchers both in academia and in the pharmaceutical industry have developed several potent and selective small molecule inhibitors of KCa3.1. This article will briefly review the available compounds (TRAM-34, senicapoc, NS6180), their binding sites and mechanisms of action, and then discuss the potential usefulness of these compounds for the treatment of brain tumors based on their brain penetration and their efficacy in reducing microglia activation in animal models of ischemic stroke and Alzheimer's disease., Conclusion: Senicapoc, which has previously been in Phase III clinical trials, would be available for repurposing, and could be used to quickly translate findings made with other KCa3.1 blocking tool compounds into clinical trials., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Glycobiology, in particular the study of carbohydrate-protein interactions and the events that follow, has become an important research focus in recent decades. To study these interactions, many assays require homogeneous glycoconjugates in suitable amounts. Their synthesis is one of the methodological challenges of glycobiology. Here, we describe a versatile, three-stage protocol for the formation of glycoconjugates from unprotected carbohydrates, including those purified from natural sources, as exemplified here by rhizobial Nod factors and exopolysaccharide fragments. The first stage is to add an oligo(ethylene glycol) linker (OEG-linker) that has a terminal triphenylmethanethiol group to the reducing end of the oligosaccharide by oxime formation catalyzed by aniline. The triphenylmethyl (trityl) tag is then removed from the linker to expose a thiol (stage 2) to allow a conjugation reaction at the thiol group (stage 3). There are many possible conjugation reactions, depending on the desired application. Examples shown in this protocol are as follows: (i) coupling of the oligosaccharide to a support for surface plasmon resonance (SPR) studies, (ii) fluorescence labeling for microscale thermophoresis (MST) or bioimaging, and (iii) biotinylation for biolayer interferometry (BLI) studies. This protocol starts from unprotected carbohydrates and provides glycoconjugates in milligram amounts in just 2 d.

Tetrathiatriaylmethyl (trityl) radicals have found wide biomedical applications as magnetic resonance probes. Trityl radicals and their derivatives are generally stable toward biological reducing agents such as glutathione (GSH) and ascorbate. We demonstrate that the triester (ET-03) and triamide (AT-03) derivatives of the Finland trityl radical exhibit unique reduction by thiols such as GSH and cysteine (Cys) to generate the corresponding trityl carbanions as evidenced by the loss of EPR signal and appearance of characteristic UV-vis absorbance at 644 nm under anaerobic conditions. The trityl carbanions can be quickly converted back to the original trityl radicals by oxygen (O 2 ) in air, thus rendering the reaction between the trityl derivative and biothiol undetectable under aerobic conditions. The reduction product of O 2 by the trityl carbanions was shown to be superoxide radical (O 2 •- ) by EPR spin-trapping. Kinetic studies showed that the reaction rate constants (k) depend on the types of both trityl radicals and thiols with the order of k ET-03/Cys (0.336 M -1 s -1 ) > k ET-03/GSH (0.070 M -1 s -1 ) > k AT-03/Cys (0.032 M -1 s -1 ) > k AT-03/GSH (0.027 M -1 s -1 ). The reactivity of trityl radicals with thiols is closely related to the para-substituents of trityl radicals as well as the pK a of the thiols and is further reflected by the rate of O 2 •- production and consumptions of O 2 and thiols. This novel reaction represents a new metabolic process of trityl derivatives and should be considered in the design and application of new trityl radical probes.

In cancer therapy, optimizing tumor-specific delivery, tumor distribution, and cellular uptake of a drug is important for ensuring minimal toxicity and maximum therapeutic efficacy. This study characterized the therapeutic efficacy of a stimulus responsive and dual targeting nanocarrier for a bioimaging-guided photothermal and chemotherapeutic platform. Hyaluronic acid (HA) conjugated with triphenylphosphonium (TPP) and boronic acid (BA) diol-linked β-cyclodextrin (β-CD) forms an inclusion complex with paclitaxel (PTX), creating a shell-like composite on a core of carbonized fluorescent polydopamine nanoparticles (FNPs-pDA) applicable for photothermal therapy as well as bioimaging. The successful diol cross-linking between core@shells generates nanocarriers [FNPs-pDA@HA-TPP-CD-PTX] that can be used as an extracellular HA- and intracellular TPP-mediated dual targeting system. The carbonized FNPs-pDA was cross-linked with the boronic acid groups of HA-TPP-CD-PTX to promote the formation of boronate esters for pH-mediated photothermal activity, which have shown time dependent complete PTX release along with a photothermal mediated response. The in vitro dual bioimaging and photothermal-chemotherapeutic activities were compared between cancer and normal cells. Lysosomal escape and live/dead cells staining confocal images highlight the promise of this system, which might open up a new approach, a simple and versatile method for site-specific synergetic drug delivery.

Superoxide radical is involved in numerous physiological and pathophysiological processes. Tetrathiatriarylmethyl (TAM) radicals are known to react with superoxide allowing measurement of superoxide production in biological media. We report the synthesis of a Nitro conjugated TAM radical showing a rate constant of 7 × 10 5  M -1  s -1 which is two order of magnitude higher than other TAMs, allowing high sensitivity measurement of superoxide.