Prebiotic RNA self-assembling and the origin of life: Mechanistic and molecular modeling rationale for explaining the prebiotic origin and replication of RNA.

In recent years, a great interest has been focused on the prebiotic origin of nucleic acids and life on Earth. An attractive idea is that life was initially based on an autocatalytic and autoreplicative RNA (the RNA-world). RNA duplexes are right-handed helical chains with antiparallel orientation, but the rationale for these features is not yet known.

How to Target Small-Molecule Fluorescent Imaging Probes to the Plasma Membrane-The Influence and QSAR Modelling of Amphiphilicity, Lipophilicity, and Flip-Flop.

Many new fluorescent probes targeting the plasma membrane (PM) of living cells are currently being described. Such probes are carefully designed to report on relevant membrane features, but oddly, the structural features required for effective and selective targeting of PM often receive less attention, constituting a lacuna in the molecular design process. We aim to rectify this by clarifying how the amphiphilicity and lipophilicity of a probe, together with the tendency to flip-flop across the membrane, contribute to selective PM accumulation.

Classification and naming of polymethine dyes used as staining agents for microscopy. A short guide for biomedical investigators.

The scientific literature contains many accounts of application of polymethine dyes, including cyanine dyes, as imaging agents, i.e., "biological stains," for microscopic investigation of biological materials.

Modeling the extent of conjugation in histochemical dyes and fluorescent probes: clarification in calculating conjugated bond number (CBN) and introduction of a new parameter, resonance energy.

Determining the extent of conjugation in dyes and fluorochromes is a helpful tool for understanding or predicting the behavior of these compounds when used as stains for microscopy. One measure that has been used repeatedly is conjugated bond number (CBN), which is the number of bonds in a conjugated system. CBN can be obtained by inspection of the structure of a compound, but the rules for how to determine what constitutes a conjugated system are not fully established.

Fluorescence labeling of mitochondria in living cells by the cationic photosensitizer ZnTM2,3PyPz, and the possible roles of redox processes and pseudobase formation in facilitating dye uptake.

The study of labeling selectivity and mechanisms of fluorescent organelle probes in living cells is of continuing interest in biomedical sciences. The tetracationic phthalocyanine-like ZnTM2,3PyPz photosensitizing dye induces a selective violet fluorescence in mitochondria of living HeLa cells under UV excitation that is due to co-localization of the red signal of the dye with NAD(P)H blue autofluorescence. Both red and blue signals co-localize with the green emission of the mitochondria probe, rhodamine 123.

At least four distinct blue cationic phthalocyanine dyes sold as "alcian blue" raises the question: what is alcian blue?

We investigated physicochemical characteristics of dye lots sold as "alcian blue" using the Biological Stain Commission (BSC) precipitation test, differential scanning calorimetry, high performance liquid chromatography, thin layer chromatography and UV/visible spectroscopy. Four blue phthalocyanine dyes were detected in 11 commercial dye lots. These four included the original ingrain blue 1 CI 74000 dye and the dye sold with the name "alcian blue pyridine variant"; we discuss also the possible identity of the additional two dyes.

Using QSAR Models to Predict Mitochondrial Targeting by Small-Molecule Xenobiotics Within Living Cells.

Prediction of mitochondrial targeting, or prediction of exclusion from mitochondria, of small-molecule xenobiotics (biocides, drugs, probes, toxins) can be achieved using an algorithm derived from QSAR modeling. Application of the algorithm requires knowing the chemical structures of all ionic species of the xenobiotic compound in question, and for certain numerical structure parameters (AI, CBN, log P, pK , and Z) to be obtained for all such species. Procedures for specification of the chemical structures; estimation of the structure parameters; and application of the algorithm are described in an explicit protocol.

Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate.

Natural and synthetic phenazines are widely used in biomedical sciences. In dehydrogenase histochemistry, phenazine methosulfate (PMS) is applied as a redox reagent for coupling reduced coenzymes to the reduction of tetrazolium salts into colored formazans. PMS is also currently used for cytotoxicity and viability assays of cell cultures using sulfonated tetrazoliums.

Revised tests and standards for Biological Stain Commission certification of alcian blue dyes.

Alcian blue dyes are copper phthalocyanines with a variety of cationic side chains; they are useful for staining carbohydrate polyanions while avoiding staining of nucleic acids. The properties of the original alcian blue and of similar dyes with published chemical structures are reviewed here. Variation among samples submitted to the Biological Stain Commission (BSC) for certification has led to the recognition of two types of commercially available alcian blue at this time.

Tetrazolium salts and formazan products in Cell Biology: Viability assessment, fluorescence imaging, and labeling perspectives.

For many years various tetrazolium salts and their formazan products have been employed in histochemistry and for assessing cell viability. For the latter application, the most widely used are 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and 5-cyano-2,3-di-(p-tolyl)-tetrazolium chloride (CTC) for viability assays of eukaryotic cells and bacteria, respectively. In these cases, the nicotinamide-adenine-dinucleotide (NAD(P)H) coenzyme and dehydrogenases from metabolically active cells reduce tetrazolium salts to strongly colored and lipophilic formazan products, which are then quantified by absorbance (MTT) or fluorescence (CTC).

A review of curcumin as a biological stain and as a self-visualizing pharmaceutical agent.

Curcumin has been widely used to color textiles but, unlike other natural dyes such as hematoxylin or saffron, it rarely has been discussed as a biological stain. Aspects of the physicochemistry of curcumin relevant to biological staining and self-visualization, i.e.

Prediction of Intracellular Localization of Fluorescent Dyes Using QSAR Models.

Control of fluorescent dye localization in live cells is crucial for fluorescence imaging. Here, we describe quantitative structure activity relation (QSAR) models for predicting intracellular localization of fluorescent dyes. For generating the QSAR models, electric charge (Z) calculated by pKa, conjugated bond number (CBN), the largest conjugated fragment (LCF), molecular weight (MW) and log P were used as parameters.

Necrosis avid near infrared fluorescent cyanines for imaging cell death and their use to monitor therapeutic efficacy in mouse tumor models.

Quantification of tumor necrosis in cancer patients is of diagnostic value as the amount of necrosis is correlated with disease prognosis and it could also be used to predict early efficacy of anti-cancer treatments. In the present study, we identified two near infrared fluorescent (NIRF) carboxylated cyanines, HQ5 and IRDye 800CW (800CW), which possess strong necrosis avidity. In vitro studies showed that both dyes selectively bind to cytoplasmic proteins of dead cells that have lost membrane integrity.

Massive Bioaccumulation and Self-Assembly of Phenazine Compounds in Live Cells.

Clofazimine is an orally administered, FDA-approved drug that massively bioaccumulates in macrophages, forming membrane-bound intracellular structures possessing nanoscale supramolecular features. Here, a library of phenazine compounds derived from clofazimine was synthesized and tested for their ability to accumulate and form ordered molecular aggregates inside cells. Regardless of chemical structure or physicochemical properties, bioaccumulation was consistently greater in macrophages than in epithelial cells.

Uptake and localization mechanisms of fluorescent and colored lipid probes. Part 2. QSAR models that predict localization of fluorescent probes used to identify ("specifically stain") various biomembranes and membranous organelles.

We discuss a variety of biological targets including generic biomembranes and the membranes of the endoplasmic reticulum, endosomes/lysosomes, Golgi body, mitochondria (outer and inner membranes) and the plasma membrane of usual fluidity. For each target, we discuss the access of probes to the target membrane, probe uptake into the membrane and the mechanism of selectivity of the probe uptake. A statement of the QSAR decision rule that describes the required physicochemical features of probes that enable selective staining also is provided, followed by comments on exceptions and limits.

Uptake and localization mechanisms of fluorescent and colored lipid probes. Part 3. Protocols for predicting intracellular localization of lipid probes using QSAR models.

We provide detailed protocols for applying the QSAR decision-rule models described in Part 2 of this paper. These procedures permit prediction of the intracellular localization of fluorescent probes or of any small molecular xenobiotic whether fluorescent or not. Also included is a set of notes that give practical advice on various possible problems and limitations of the methods, together with a flow chart that provides a graphical algorithmic summary of the QSAR models.

Predicting mitochondrial targeting by small molecule xenobiotics within living cells using QSAR models.

Whether small molecule xenobiotics (biocides, drugs, probes, toxins) will target mitochondria in living cells can be predicted using an algorithm derived from QSAR modeling. Application of the algorithm requires the chemical structures of all ionic species of the xenobiotic compound in question to be defined, and for certain numerical structure parameters (AI, CBN, log P, pKa, and Z) to be obtained for all such species. How the chemical structures are specified, how the structure parameters are obtained or estimated, and how the algorithm is used are described in an explicit protocol.

News from the Biological Stain Commission no. 15.

In the 15(th) issue of News from the Biological Stain Commission (BSC), under the heading of Regulatory affairs, the Biological Stain Commission's International Affairs Committee presents information from the plenary meetings of the International Standards Organization ISO/TC 212 Clinical laboratory testing and in vitro diagnostic test systems held on August 22-24, 2012 in Berlin, Germany. An additional discussion of the use of food dyes in India also is included.

Identifying different types of chromatin using Giemsa staining.

Mixtures of polychrome methylene blue-eosin Y (i.e., Giemsa stain) are widely used in biological staining.

Alternative methods for estimating common descriptors for QSAR studies of dyes and fluorescent probes using molecular modeling software. 2. Correlations between log P and the hydrophilic/lipophilic index, and new methods for estimating degrees of amphiphilicity.

The log P descriptor, despite its usefulness, can be difficult to use, especially for researchers lacking skills in physical chemistry. Moreover this classic measure has been determined in numerous ways, which can result in inconsistant estimates of log P values, especially for relatively complex molecules such as fluorescent probes. Novel measures of hydrophilicity/lipophilicity (the Hydrophilic/Lipophilic Index, HLI) and amphiphilicity (hydrophilic/lipophilic indices for the head group and tail, HLIT and HLIHG, respectively) therefore have been devised.

Predicting small molecule fluorescent probe localization in living cells using QSAR modeling. 2. Specifying probe, protocol and cell factors; selecting QSAR models; predicting entry and localization.

We describe the practical issues and the methodological procedures that must be carried out to construct and use QSAR models for predicting localization of probes in single cells. We address first the determination of probe factors starting with a consideration of the chemical nature of probe molecules present. What is their identity? Do new compounds arise in incubation media or intracellularly? For each probe, how many distinct chemical species are present? For each probe species, the derivation of the following numerical structure parameters, or descriptors, is set out with worked examples of electric charge and acid/base strength (Z and pKa); hydrophilicity/lipophilicity (log P); amphiphilicity (AI and HGH); conjugated bond number and largest conjugated fragment (CBN and LCF); width and length (W and L); and molecular and ionic weights, head group size and substituent bulk (MW, IW, HGS and SB).

Predicting small molecule fluorescent probe localization in living cells using QSAR modeling. 1. Overview and models for probes of structure, properties and function in single cells.

Small molecule fluorochromes (synonyms: biosensors, chemosensors, fluorescent probes, vital stains) are widely used to investigate the structure, composition, physicochemical properties and biological functions of living cells, tissues and organisms. Selective entry and accumulation within particular cells and cellular structures are key processes for achieving these diverse objectives. Despite the complexities, probes routinely are applied using standard protocols, often without experimenter awareness of what factors that control accumulation and localization.

Uptake and localisation of small-molecule fluorescent probes in living cells: a critical appraisal of QSAR models and a case study concerning probes for DNA and RNA.

Small-molecule fluorochromes are used in biology and medicine to generate informative microscopic and macroscopic images, permitting identification of cell structures, measurement of physiological/physicochemical properties, assessment of biological functions and assay of chemical components. Modes of uptake and precise intracellular localisation of a probe are typically significant factors in its successful application. These processes and localisations can be predicted using quantitative structure activity relations (QSAR) models, which correlate aspects of the physicochemical properties of the probes (expressed numerically) with the uptake/localisation.