Real-time quantification of protein expression and translocation at individual cell resolution using imaging-dish-based live cell array

Cell populations represent intrinsically heterogeneous systems with a high level of spatiotemporal complexity. Monitoring and understanding cell-to-cell diversity is essential for the research and application of intra- and interpopulation variations. Optical analysis of live cells is challenging since both adherent and nonadherent cells change their spatial location. However, most currently available single-cell techniques do not facilitate treatment and monitoring of the same live cells over time throughout multistep experiments. An imaging-dish-based live cell array (ID-LCA) has been developed and produced for cell handling, culturing, and imaging of numerous live cells. The dish is composed of an array of pico scale cavities—pico wells (PWs) embossed on its glass bottom. Cells are seeded, cultured, treated, and spatiotemporally measured on the ID-LCA, while each cell or small group of cells are locally constrained in the PWs. Finally, predefined cells can be retrieved for further evaluation. Various types of ID-LCAs were used in this proof-of-principle work, to demonstrate on-ID-LCA transfection of fluorescently tagged chimeric proteins, as well as the detection and kinetic analysis of their induced translocation. High variability was evident within cell populations with regard to protein expression levels as well as the extent and dynamics of protein redistribution. The association of these parameters with cell morphology and functional parameters was examined. Both the new methodology and the device facilitate research of the translocation process at individual cell resolution within large populations and thus, can potentially be used in high-throughput fashion. Graphical Abstract

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Potentiometric determination of the 'formal' hydrolysis ratio of aluminium species in aqueous solutions

The ‘formal’ hydrolysis ratio (h = C(OH⁻)_added/C(Al)_total) of hydrolysed aluminium-ions is an important parameter required for the exhaustive and quantitative speciation-fractionation of aluminium in aqueous solutions. This paper describes a potentiometric method for determination of the formal hydrolysis ratio based on an automated alkaline titration procedure. The method uses the point of precipitation of aluminium hydroxide as a reference (h = 3.0) in order to calculate the initial formal hydrolysis ratio of hydrolysed aluminium-ion solutions. Several solutions of pure hydrolytic species including aluminium monomers (AlCl₃), Al₁₃ polynuclear cluster ([Al₁₃O₄(OH)₂₄(H₂O)₁₂]⁷⁺), Al₃₀ polynuclear cluster ([Al₃₀O₈(OH)₅₆(H₂O)₂₆]¹⁸⁺) and a suspension of nanoparticulate aluminium hydroxide have been used as ‘reference standards’ to validate the proposed potentiometric method. Other important variables in the potentiometric determination of the hydrolysis ratio have also been optimised including the concentration of aluminium and the type and strength of alkali (Trizma-base, NH₃, NaHCO₃, Na₂CO₃ and KOH). The results of the potentiometric analysis have been cross-verified by quantitative ²⁷Al solution nuclear magnetic resonance (²⁷Al NMR) measurements. The ‘formal’ hydrolysis ratio of a commercial basic aluminium chloride has been measured as an example of a practical application of the developed technique.     

Progress in intermolecular and intramolecular reactions of thioamides with diazo compounds and azides

Reactions of thioamides with nitrogen-rich 1,3-dipoles, diazo compounds and azides, have been known for long time already. However in recent years introduction of catalysts of different types (rhodium-, ruthenium- and copper-containing and Lewis acids) as well as highly electrophilic sulfonyl azides, allowed the development of new methods for the synthesis of heterocycles, enamines and N-sulfonyl amidines. Moreover, a new methodology in organic synthesis, based on generation and subsequent transformations of α-diazocarbonyl compounds was created. Reactions of sulfonyl azides with thioamides undergo readily in mild conditions to produce different sorts of N-sulfonyl amidines and represent a new type of click-type processes. Most of the cited works were published in the current decade. Earlier seminal papers are also reviewed when they constitute the background for new synthetic methods which were developed further.     

β-(Cycloalkylamino)ethanesulfonyl azides as novel water-soluble reagents for the synthesis of diazo compounds and heterocycles

Chemistry of Heterocyclic Compounds - Novel water-soluble sulfonyl azides were synthesized, which are basic by nature. The obtained compounds were used as donors of the diazo group in the diazo...     

Mass-spectrometric study of the cyclization of diazo compounds. 9. 2-Diazo-2-cyanoacetamides

An analysis of the electron impact mass-spectra of 2-diazo-2-cyanoacetamides and the 4-cyano-5-hydroxy-1,2,3-triazoles isomeric to them, showed that the molecular ions of these compounds do not isomerize one into another. The diazo compounds decompose, undergoing a Wolff rearrangement. To study the fragmentation of the diazoamides, one can use the crystalline adducts of these diazo compounds with triphenylphosphine, and to study the fragmentation of the triazoles, their salts with aliphatic amines.See [1] for No. 8 in the series.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 10, pp. 1343–1349, October, 1986.     

Heterocyclization of compounds containing diazo and cyano groups. 2. Synthesis and recyclization of 4-substituted 5-amino-1,2,3-thiadiazoles

The reaction of carbonyl derivatives of diazoacetonitrile with hydrogen sulfide in the presence of triethylamine yields 4-substituted 5-amino-1,2,3-triadiazoles. Under analogous conditions, hydrogen selenide and ethyl mercaptan reduce the starting diazo compounds to hydrazones. Thiadiazoles are recyclized to 4-substituted 5-mercapto-1,2,3-triazoles by the action of bases.For 1, see [1].Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 5, pp. 691–696, May, 1986.     

Anomalous rearrangement of 1,2,3-thiadiazoles to 1,2,3-triazoles

The rearrangement of 5-amino-1,2,3-thiadiazoles under the influence of halogen-containing oxidizing to bis(triazolyl) disulfides was observed. Ammonia reduces the disulfides obtained to 5-mercapto-1,2,3-triazoles.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 6, pp. 840–846, June, 1993.