Covalently Modified Molecular-Recognition-Capable UV-Transparent Microplate for Ultra-High-Throughput Screening of Dissolved Zn2+ and Pb2.

Zn2+ has a crucial role both in biology and the environment, while Pb2+ presents serious hazards in the same areas due to its toxicity, and the need for their analysis often exceeds available instrumental capacity. We report, herein, a new high-throughput optochemical screening method for Zn2+ and Pb2+ in various solutions. Moreover, we also introduced a new and generalizable three-step-microplate-modification technique, including plasma treating, linker-docking and photocatalytic copolymerization. The surface of a commercially available 96-well-cycloolefin-microplate was treated with atmospheric plasma, and then, the bottoms of the wells were covered by covalently attaching a methacrylate-containing linker-monolayer. Finally, the preactivated microplate wells were covalently functionalized by immobilizing bis(acridino)-crown ether-type sensor molecules, via photocatalytic copolymerization, to a polymethacrylate backbone. This sensing tool can be used in all microplate readers, is compatible with liquid handling platforms and provides an unprecedently fast monitoring (>1000 samples/hour, extrapolated from the time required for 96 measurements) of dissolved Zn2+ and Pb2+ among recent alternatives above the detection limits of 8.0 × 10-9 and 3.0 × 10-8 mol/L, respectively, while requiring a sample volume of only 20 µL.

Synthetic Modifications of a Pb2+-Sensor Acridono-Crown Ether for Covalent Attachment and Their Effects on Selectivity.

Because of environmental impact, there is a great need for chemosensors, especially for toxic heavy metals such as lead. The conventional instrumental analytical techniques rarely provide an available real-time sensing platform, thus the development of highly selective and stable synthetic chemosensor molecules is of great importance. Acridono-18-crown-6 ethers have such properties, and much research has proven their outstanding applicability in various supramolecular devices. In this present work, we aimed to enable their covalent immobilization capability by synthesizing functionalized derivatives while preserving the favored molecular recognition ability. Several new macrocycle analogues were synthesized, while synthetization difficulties and design aspects were also dealt with. The selectivity of the macrocycle analogues was studied using UV-Vis spectroscopy and compared with that of the parent compounds. The ultimate crown ether derivative showed high Pb2+-selectivity, reversibility (decomplexation by extraction with water) and stability.

New Polymerizable Tetraaza Macrocycle Containing Two Acridine Units for Selective Fluorescence Sensing of Metal Ions.

A new fluorescent bis(acridino)-macrocycle containing two allyl groups was synthesized and photophysically studied. Studies were carried out on metal ion recognition and selectivity-influencing effects including the determination of the relevant thermodynamic constants as logK and pKa. The proposed sensor molecule is recommended for the development of Zn2+-selective optochemical analyzers based on covalently immobilized ionophores as it has a unique pH-independent metal ion recognition ability, which is not influenced by anions and other potentially occurring metal ions in biological samples.

B cell receptor ligation induces display of V-region peptides on MHC class II molecules to T cells.

The B cell receptors (BCRs) for antigen express variable (V) regions that are enormously diverse, thus serving as markers on individual B cells. V region-derived idiotypic (Id) peptides can be displayed as pId:MHCII complexes on B cells for recognition by CD4+ T cells. It is not known if naive B cells spontaneously display pId:MHCII in vivo or if BCR ligation is required for expression, thereby enabling collaboration between Id+ B cells and Id-specific T cells. Here, using a mouse model, we show that naive B cells do not express readily detectable levels of pId:MHCII. However, BCR ligation by Ag dramatically increases physical display of pId:MHCII, leading to activation of Id-specific CD4+ T cells, extrafollicular T-B cell collaboration and some germinal center formation, and production of Id+ IgG. Besides having implications for immune regulation, the results may explain how persistent activation of self-reactive B cells induces the development of autoimmune diseases and B cell lymphomas.

Covalently Immobilizable Tris(Pyridino)-Crown Ether for Separation of Amines Based on Their Degree of Substitution.

A great number of biologically active compounds contain at least one amine function. Appropriate selectivity can only be accomplished in a few cases upon the substitution of these groups, thus functionalization of amines generally results in a mixture of them. The separation of these derivatives with very similar characteristics can only be performed on a preparative scale or by applying pre-optimized HPLC methods. A tris(pyridino)-crown ether was designed and synthetized for overcoming these limitations at a molecular level. It is demonstrated, that this selector molecule is able to distinguish protonated primary, secondary and tertiary amines by the formation of reversible complexes with different stabilities. This degree of substitution-specific molecular recognition of amines opens the door to develop separation processes primarily focusing on the purification of biologically active compounds in a nanomolar scale.

Acridino-Diaza-20-Crown-6 Ethers: New Macrocyclic Hosts for Optochemical Metal Ion Sensing.

Acridino-diaza-20-crown-6 ether derivatives as new turn-on type fluorescent chemosensors with an excellent functionality and photophysical properties have been designed and synthesized for metal ion-selective optochemical sensing applications. Spectroscopic studies revealed that in an acetonitrile-based semi-aqueous medium, the sensor molecules exhibited a remarkable fluorescence enhancement with high sensitivity only toward Zn2+, Al3+ and Bi3+, among 23 different metal ions. Studies on complexation showed a great coordinating ability of logK > 4.7 with a 1:1 complex stoichiometry in each case. The detection limits were found to be from 59 nM to micromoles. The new ionophores enabled an optical response without being affected either by the pH in the range of 5.5-7.5, or the presence of various anions or competing metal ions. Varying the N-substituents of the new host-backbone provides diverse opportunities in both immobilization and practical applications without influencing the molecular recognition abilities.

Synthesis of Novel Crown Ether-Squaramides and Their Application as Phase-Transfer Catalysts.

This work presents the synthesis of six new phase-transfer organocatalysts in which the squaramide unit is directly linked to the nitrogen atom of an aza-crown ether. Four chiral skeletons, namely hydroquinine, quinine, cinchonine (cinchonas), and α-d-glucopyranoside were responsible for the asymmetric construction of an all-carbon quaternary stereogenic center in α-alkylation and Michael addition reactions of malonic esters. We investigated the effects of these different chiral units and that of crown ethers with different sizes on catalytic activity and enantioselectivity. During extensive parameter investigations, both conventional and emerging green solvents were screened, providing valuable α,α-disubstituted malonic ester derivatives with excellent yields (up to 98%).

Synthesis and Applications of Cinchona Squaramide-Modified Poly(Glycidyl Methacrylate) Microspheres as Recyclable Polymer-Grafted Enantioselective Organocatalysts.

This work presents the immobilization of cinchona squaramide organocatalysts on poly(glycidyl methacrylate) solid supports. Preparation of the well-defined monodisperse polymer microspheres was facilitated by comprehensive parameter optimization. By exploiting the reactive epoxy groups of the polymer support, three amino-functionalized cinchona derivatives were immobilized on this carrier. To explore the effect of the amino linker, these structurally varied precatalysts were synthesized by modifying the cinchona skeleton at different positions. The catalytic activities of the immobilized organocatalysts were tested in the Michael addition of pentane-2,4-dione and trans-ß-nitrostyrene with excellent yields (up to 98 %) and enantioselectivities (up to 96 % ee). Finally, the catalysts were easily recovered five times by centrifugation without loss of activity.

Synthesis, Molecular Recognition Study and Liquid Membrane-Based Applications of Highly Lipophilic Enantiopure Acridino-Crown Ethers.

New highly lipophilic enantiopure crown ethers containing a heterocyclic unit have been synthesized. Phase transport, UV-Vis- and fluorescence spectrophotometric investigations as well as electrochemical studies on the complexation of the new macrocycles with several amine and amino acid derivatives were also carried out. Achiral amines were used for studying the structural preference of the new macrocycles. Among the studied structural features of the guest molecules, the intermolecular π-π interaction showed the most significant effect on complexation, which made the aralkylamine-type compounds the most preferable guest molecules. The studied liquid membrane-based applications and photophysical investigations showed appreciable enantiomeric recognition toward some aralkylamine model compounds with homochiral preferences. New crown ether derivatives (R,R)-2 and (S,S)-2 were successfully applied as enantioselective carrier and sensor molecules.

Brain-resident memory CD8+ T cells induced by congenital CMV infection prevent brain pathology and virus reactivation.

Congenital HCMV infection is a leading infectious cause of long-term neurodevelopmental sequelae. Infection of newborn mice with mouse cytomegalovirus (MCMV) intraperitoneally is a well-established model of congenital human cytomegalovirus infection, which best recapitulates the hematogenous route of virus spread to brain and subsequent pathology. Here, we used this model to investigate the role, dynamics, and phenotype of CD8+ T cells in the brain following infection of newborn mice. We show that CD8+ T cells infiltrate the brain and form a pool of tissue-resident memory T cells (TRM cells) that persist for lifetime. Adoptively transferred virus-specific CD8+ T cells provide protection against primary MCMV infection in newborn mice, reduce brain pathology, and remain in the brain as TRM cells. Brain CD8+ TRM cells were long-lived, slowly proliferating cells able to respond to local challenge infection. Importantly, brain CD8+ TRM cells controlled latent MCMV and their depletion resulted in virus reactivation and enhanced inflammation in brain.

Optically active crown ether-based fluorescent sensor molecules: A mini-review.

This mini-review focuses on fluorescent optically active crown ethers (polymeric derivatives are not included) reported in the literature (according to our knowledge), of which enantiomeric recognition ability, and in some cases, also inorganic cation complexation properties, were investigated by the sensitive and versatile fluorescence spectroscopy. These crown ether-based chemosensors contain various fluorophore signaling units such as binaphthyl, anthracene, pyrene, tryptophan, benzimidazole, terpyridine, acridine, phenazine, acridone, BODIPY, and another conjugated aromatic one.

Biomimetic Synthesis of Drug Metabolites in Batch and Continuous-Flow Reactors.

A medium-throughput screening (MTS) of biomimetic drug metabolite synthesis is developed by using an iron porphyrin catalyst. The microplate method, in combination with HPLC-MS analysis, was shown to be a useful tool for process development and parameter optimization in the production of targeted metabolites and/or oxidation products of forty-three different drug substances. In the case of the biomimetic oxidation of amiodarone, the high quantity and purity of the isolated products enabled detailed HRMS and NMR spectroscopic studies. In addition to identification of known metabolites, several new oxidation products of the drug that was studied were characterized. Fast degradation and poor recovery of the catalyst under batch conditions was overcome by immobilization of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin iron(III) chloride (FeTSPP) on the surface of 3-aminopropyl-functionalized silica by electrostatic interaction. The supported catalyst was successfully applied in a packed-bed reactor under continuous-flow reaction conditions for the large-scale synthesis of amiodarone metabolites.

Cathepsin L silencing increases As2O3 toxicity in malignantly transformed pilocytic astrocytoma MPA58 cells by activating caspases 3/7.

Low-grade, pilocytic astrocytomas are treated by resection, but additional therapy is necessary for those tumors with anaplastic features. Arsenic trioxide (As2O3) is emerging as an effective chemotherapeutic agent for treatment of malignant glioblastoma multiforme, where Cathepsin L silencing enables lower, less harmful As2O3 concentrations to achieve the desired cytotoxic effect. Here, we evaluated the effects of As2O3 combined with stable Cathepsin L shRNA silencing on cell viability/metabolic activity, and apoptosis in primary cultures of recurrent malignantly transformed pilocytic astrocytoma (MPA). These cells expressed high Cathepsin L levels, and when grown as monolayers and spheroids, they were more resistant to As2O3 than the U87MG glioblastoma cell line. Caspases 3/7 activity in MPA58 spheroids was not significantly affected by As2O3, possibly due to higher chemoresistance of primary biopsy tissue of less malignant astrocytoma versus the malignant U87MG cell line. However, As2O3 treatment was cytotoxic to MPA spheroids after silencing of Cathepsin L expression. While Cathepsin L silencing only slightly decreased the live/dead cell ratio in As2O3-treated MPA-si spheroids under our experimental conditions, there was an increase in As2O3-mediated apoptosis in MPA-si spheroids, as indicated by elevated caspases 3/7 activity. Therefore, Cathepsin L silencing by gene manipulation can be applied when a more aggressive approach is needed in treatment of pilocytic astrocytomas with anaplastic features.

Fast Potentiometric Analysis of Lead in Aqueous Medium under Competitive Conditions Using an Acridono-Crown Ether Neutral Ionophore.

Lead is a particularly toxic heavy metal that is present above acceptable levels in the water of many countries. This article describes a quick detection method of lead(II) ions using a polyvinyl chloride (PVC)-based ion-selective membrane electrode containing an acridono-crown ether ionophore by potentiometry. The electrochemical cell exhibits a Nernstian response for lead(II) ions between the concentration range of 10−4 to 10−2 M, and can be used in the pH range of 4⁻7. The applicability of this sensor was verified by measuring a multicomponent aqueous sample. Under the given conditions, this electrode is suitable for the selective quantitative analysis of lead(II) ions in the presence of many additional metal ions.

Synthesis and pKa determination of new enantiopure dimethyl-substituted acridino-crown ethers containing a carboxyl group: Useful candidates for enantiomeric recognition studies.

New enantiopure dimethyl-substituted acridino-18-crown-6 and acridino-21-crown-7 ethers containing a carboxyl group at position 9 of the acridine ring [(S,S)-8, (S,S)-9, (R,R)-10] were synthesized. The pKa values of the new crown ethers [(S,S)-8, (S,S)-9, (R,R)-10] and of an earlier reported macrocycle [(R,R)-2] were determined by UV-pH titrations. Crown ether (S,S)-8 was attached to silica gel by covalent bonds and the enantiomeric separation ability of the newly prepared chiral stationary phase [(S,S)-CSP-12] was studied by high-performance liquid chromatography (HPLC). Homochiral preference was observed and the best separation was achieved for the enantiomers of 1-NEA. Ligands (S,S)-9 and (R,R)-10 are precursors of enantioselective sensor and selector molecules for the enantiomers of protonated primary amines, amino acids, and their derivatives.

Synthesis and Enantiomeric Recognition Studies of Optically Active Pyridino-Crown Ethers Containing an Anthracene Fluorophore Unit.

Novel enantiopure pyridino-18-crown-6 ether-based sensor molecules containing an anthracene fluorophore unit were synthesized. Their enantiomeric recognition abilities toward the enantiomers of 1-phenylethylamine hydrogen perchlorate (PhEt), 1-(1-naphthyl)ethylamine hydrogen perchlorate (NapEt), phenylglycine methyl ester hydrogen perchlorate (PhgOMe), and phenylalanine methyl ester hydrogen perchlorate (PheOMe) were examined in acetonitrile using fluorescence spectroscopy. The sensor molecules showed appreciable enantiomeric recognition toward the enantiomers of NapEt, PhEt, and PhgOMe. The highest enantioselectivity was found in the case of crown ether containing isobutyl groups in the macroring and the enantiomers of NapEt. Chirality 28:562-568, 2016. © 2016 Wiley Periodicals, Inc.

Selected Aspects in the Pathogenesis of Autoimmune Diseases.

Autoimmune processes can be found in physiological circumstances. However, they are quenched with properly functioning regulatory mechanisms and do not evolve into full-blown autoimmune diseases. Once developed, autoimmune diseases are characterized by signature clinical features, accompanied by sustained cellular and/or humoral immunological abnormalities. Genetic, environmental, and hormonal defects, as well as a quantitative and qualitative impairment of immunoregulatory functions, have been shown in parallel to the relative dominance of proinflammatory Th17 cells in many of these diseases. In this review we focus on the derailed balance between regulatory and Th17 cells in the pathogenesis of autoimmune diseases. Additionally, we depict a cytokine imbalance, which gives rise to a biased T-cell homeostasis. The assessment of Th17/Treg-cell ratio and the simultaneous quantitation of cytokines, may give a useful diagnostic tool in autoimmune diseases. We also depict the multifaceted role of dendritic cells, serving as antigen presenting cells, contributing to the development of the pathognomonic cytokine signature and promote cellular and humoral autoimmune responses. Finally we describe the function and role of extracellular vesicles in particular autoimmune diseases. Targeting these key players of disease progression in patients with autoimmune diseases by immunomodulating therapy may be beneficial in future therapeutic strategies.

Cathepsin L silencing enhances arsenic trioxide mediated in vitro cytotoxicity and apoptosis in glioblastoma U87MG spheroids.

Despite improved treatment options, glioblastoma multiforme (GBM) remains the most aggressive brain tumour with the shortest post-diagnostic survival. Arsenite (As2O3) is already being used in the treatment of acute promyelocytic leukaemia (APL), yet its effects on GBM have not been evaluated in detail. In U87MG cell monolayers, we have previously shown that arsenite cytotoxicity significantly increases upon transient inhibition of lysosomal protease Cathepsin L (CatL). As multicellular spheroids more closely represent in vivo tumours, we aimed to evaluate the impact of permanent CatL silencing on arsenite treatment in U87MG spheroids. CatL was stably silenced using shRNA expression plasmid packed lentiviruses. By using metabolic- and cell viability assays, we demonstrated that long-term CatL silencing significantly increased arsenite cytotoxicity in U87MG spheroids. Silenced CatL also increased arsenite-mediated apoptosis in spheroids via elevated p53 expression, Bax/Bcl2 ratio and caspase 3/7 activity, though with lower efficacy than in monolayers. Arsenite cytotoxicity was enhanced by lower CatL activity, since similar cytotoxicity increase was also observed using the novel CatL inhibitor AT094. The results have significant translational impact, since stable CatL silencing would enable the application of lower systemic doses of arsenite to achieve the desired cytotoxic effects on GBMs in vivo.

Preparation and studies of chiral stationary phases containing enantiopure acridino-18-crown-6 ether selectors.

The enantiomeric separation ability of the newly prepared chiral stationary phases containing acridino-18-crown-6 ether selectors was studied by high-performance liquid chromatography (HPLC). The chiral stationary phases separated the enantiomers of selected protonated primary aralkylamines efficiently. The best results were found for the separation of the mixtures of enantiomers of NO2 -PEA.

Ultrafast Solid-Phase Oxidation of Aldehydes to Carboxylic Acids by Atmosphseric Plasma Treatment.

Although atmospheric plasma treatment is an industrially widespread, scalable, and environmentally friendly method, it has been generally used for surface modification, decontamination, or sterilization. In this paper, a novel, sustainable, green, and ultrafast oxidation method is described for aldehydes on a preparative thin-layer chromatographic plate as a solid support. The plasma treatment has proven to be suitable for producing the corresponding carboxylic acids by using only air as a reactant source under mild reaction conditions, while the isolation of the products is also directly integrated into the oxidation process. Extensibility to other reaction types is not explored yet, but we are sure that this novel synthesis conception carries a lot of possibilities.