Quirks of dye nomenclature. 9. Fluorescein

Adolf Baeyer announced the discovery of fluorescein in 1871 and named it after its most striking property, i.e., fluorescence. I describe here the synthesis of fluorescein. There are seven molecular species in both the solid state or in solution. I also summarize some of the diverse applications of the dye, both medical and nonmedical, which depend mostly on the facile detection of fluorescein at low concentration. Both animal and human toxicity are examined.     

Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases

Expression of foreign pathways often results in suboptimal performance due to unintended factors such as introduction of toxic metabolites, cofactor imbalances or poor expression of pathway components. In this study we report a 120% improvement in the production of the isoprenoid-derived sesquiterpene, amorphadiene, produced by an engineered strain of Escherichia coli developed to express the native seven-gene mevalonate pathway from Saccharomyces cerevisiae (Martin et al. 2003). This substantial improvement was made by varying only a single component of the pathway (HMG-CoA reductase) and subsequent host optimization to improve cofactor availability. We characterized and tested five variant HMG-CoA reductases obtained from publicly available genome databases with differing kinetic properties and cofactor requirements. The results of our in vitro and in vivo analyses of these enzymes implicate substrate inhibition of mevalonate kinase as an important factor in optimization of the engineered mevalonate pathway. Consequently, the NADH-dependent HMG-CoA reductase from Delftia acidovorans, which appeared to have the optimal kinetic parameters to balance HMG-CoA levels below the cellular toxicity threshold of E. coli and those of mevalonate below inhibitory concentrations for mevalonate kinase, was identified as the best producer for amorphadiene (54% improvement over the native pathway enzyme, resulting in 2.5 mM or 520 mg/L of amorphadiene after 48 h). We further enhanced performance of the strain bearing the D. acidovorans HMG-CoA reductase by increasing the intracellular levels of its preferred cofactor (NADH) using a NAD⁺-dependent formate dehydrogenase from Candida boidinii, along with formate supplementation. This resulted in an overall improvement of the system by 120% resulting in 3.5 mM or 700 mg/L amorphadiene after 48 h of fermentation. This comprehensive study incorporated analysis of several key parameters for metabolic design such as in vitro and in vivo kinetic performance of variant enzymes, intracellular levels of protein expression, in-pathway substrate inhibition and cofactor management to enable the observed improvements. These metrics may be applied to a broad range of heterologous pathways for improving the production of biologically derived compounds.     

Application of Artificial Intelligence/Machine Vision & Learning for the Development of a Live Single-cell Phenotypic Biomarker Test to Predict Prostate Cancer Tumor Aggressiveness

To assess the usefulness and applications of machine vision (MV) and machine learning (ML) techniques that have been used to develop a single cell-based phenotypic (live and fixed biomarkers) platform that correlates with tumor biological aggressiveness and risk stratification, 100 fresh prostate samples were acquired, and areas of prostate cancer were determined by post-surgery pathology reports logged by an independent pathologist. The prostate samples were dissociated into single-cell suspensions in the presence of an extracellular matrix formulation. These samples were analyzed via live-cell microscopy. Dynamic and fixed phenotypic biomarkers per cell were quantified using objective MV software and ML algorithms. The predictive nature of the ML algorithms was developed in two stages. First, random forest (RF) algorithms were developed using 70% of the samples. The developed algorithms were then tested for their predictive performance using the blinded test dataset that contained 30% of the samples in the second stage. Based on the ROC (receiver operating characteristic) curve analysis, thresholds were set to maximize both sensitivity and specificity. We determined the sensitivity and specificity of the assay by comparing the algorithm-generated predictions with adverse pathologic features in the radical prostatectomy (RP) specimens. Using MV and ML algorithms, the biomarkers predictive of adverse pathology at RP were ranked and a prostate cancer patient risk stratification test was developed that distinguishes patients based on surgical adverse pathology features. The ability to identify and track large numbers of individual cells over the length of the microscopy experimental monitoring cycles, in an automated way, created a large biomarker dataset of primary biomarkers. This biomarker dataset was then interrogated with ML algorithms used to correlate with post-surgical adverse pathology findings. Algorithms were generated that predicted adverse pathology with >0.85 sensitivity and specificity and an AUC (area under the curve) of >0.85. Phenotypic biomarkers provide cellular and molecular details that are informative for predicting post-surgical adverse pathologies when considering tumor biopsy samples. Artificial intelligence ML-based approaches for cancer risk stratification are emerging as important and powerful tools to compliment current measures of risk stratification. These techniques have capabilities to address tumor heterogeneity and the molecular complexity of prostate cancer. Specifically, the phenotypic test is a novel example of leveraging biomarkers and advances in MV and ML for developing a powerful prognostic and risk-stratification tool for prostate cancer patients.     

ALS/FTD‐associated FUS activates GSK‐3β to disrupt the VAPB–PTPIP51 interaction and ER–mitochondria associations

Defective FUS metabolism is strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), but the mechanisms linking FUS to disease are not properly understood. However, many of the functions disrupted in ALS/FTD are regulated by signalling between the endoplasmic reticulum (ER) and mitochondria. This signalling is facilitated by close physical associations between the two organelles that are mediated by binding of the integral ER protein VAPB to the outer mitochondrial membrane protein PTPIP51, which act as molecular scaffolds to tether the two organelles. Here, we show that FUS disrupts the VAPB–PTPIP51 interaction and ER–mitochondria associations. These disruptions are accompanied by perturbation of Ca²⁺ uptake by mitochondria following its release from ER stores, which is a physiological read‐out of ER–mitochondria contacts. We also demonstrate that mitochondrial ATP production is impaired in FUS‐expressing cells; mitochondrial ATP production is linked to Ca²⁺ levels. Finally, we demonstrate that the FUS‐induced reductions to ER–mitochondria associations and are linked to activation of glycogen synthase kinase‐3β (GSK‐3β), a kinase already strongly associated with ALS/FTD.     

Prediction and course of symptoms and lung function around an exacerbation in chronic obstructive pulmonary disease

Background

Frequent exacerbations induce a high burden to Chronic Obstructive Pulmonary Disease (COPD). We investigated the course of exacerbations in the published COSMIC study that investigated the effects of 1-year withdrawal of fluticasone after a 3-month run-in treatment period with salmeterol/fluticasone in patients with COPD.

Methods

In 373 patients, we evaluated diary cards for symptoms, Peak Expiratory Flow (PEF), and salbutamol use and assessed their course during exacerbations.

Results

There were 492 exacerbations in 224 patients. The level of symptoms of cough, sputum, dyspnea and nocturnal awakening steadily increased from 2 weeks prior to exacerbation, with a sharp rise during the last week. Symptoms of cough, sputum, and dyspnea reverted to baseline values at different rates (after 4, 4, and 7 weeks respectively), whereas symptoms of nocturnal awakening were still increased after eight weeks. The course of symptoms was similar around a first and second exacerbation. Increases in symptoms and salbutamol use and decreases in PEF were associated with a higher risk to develop an exacerbation, but with moderate predictive values, the areas under the receiver operating curves ranging from 0.63 to 0.70.

Conclusions

Exacerbations of COPD are associated with increased symptoms that persist for weeks and the course is very similar between a first and second exacerbation. COPD exacerbations are preceded by increased symptoms and salbutamol use and lower PEF, yet predictive values are too low to warrant daily use in clinical practice.     

Quirks of dye nomenclature. 1. Evans blue

The history, origin, identity, chemistry and use of Evans blue dye are described along with the first application to staining by Herbert McLean Evans in 1914. In the 1930s, the dye was marketed under the name, Evans blue dye, which was profoundly more acceptable than the ponderous chemical name.     

A covariotide model explains apparent phylogenetic structure of oxygenic photosynthetic lineages

The aims of the work were (1) to develop statistical tests to identify whether substitution takes place under a covariotide model in sequences used for phylogenetic inference and (2) to determine the influence of covariotide substitution on phylogenetic trees inferred for photosynthetic and other organisms. (Covariotide and covarion models are ones in which sites that are variable in some parts of the underlying tree are invariable in others and vice versa.) Two tests were developed. The first was a contingency test, and the second was an inequality test comparing the expected number of variable sites in two groups with the observed number. Application of these tests to 16S rDNA and tufA sequences from a range of nonphotosynthetic prokaryotes and oxygenic photosynthetic prokaryotes and eukaryotes suggests the occurrence of a covariotide mechanism. The degree of support for partitioning of taxa in reconstructed trees involving these organisms was determined in the presence or absence of sites showing particular substitution patterns. This analysis showed that the support for splits between (1) photosynthetic eukaryotes and prokaryotes and (2) photosynthetic and nonphotosynthetic organisms could be accounted for by patterns arising from covariotide substitution. We show that the additional problem of compositional bias in sequence data needs to be considered in the context of patterns of covariotide/covarion substitution. We argue that while covariotide or covarion substitution may give rise to phylogenetically informative patterns in sequence data, this may not always be so.      

Relation between the structure of benzphetamine analogues and their binding properties to cytochrome P-450 LM2

Twelve substrates of a homologous series of tertiary amines (type I substrates) have been reacted with cytochrome P-450 LM2 incorporated into unilamellar liposomes and in soluble form. The apparent spectral dissociation constants (Ks) of the substrate enzyme complexes and the induced high-spin shifts have been correlated with the electron density of distinct carbon atoms as monitored by 13C-NMR chemical shifts, the solubility of the amines and steric parameters of the substrate molecules. The results obtained led to the conclusion that two different intrinsic properties of the substrates can be discriminated in relation to the substrate-enzyme interaction. A diminished electron density at the nitrogen atom is accompanied by an increased binding affinity. The steric structure of the respective substrate determines its capability to shift the spin equilibrium to the high-spin state. Some characteristics of the active center of the enzyme are derived from the evidenced properties of the substrates.     

Turmeric: old spice,new spice

The history of chemical investigations into the yellow components of turmeric can be traced from 1815. Although the major yellow component of turmeric, curcumin, often is represented as a 1,3-diketone in the solid state and in nonaqueous solution, it exists in the enol form. The struggle to identify the chemical structure of curcumin continued for nearly a century and was complicated by the difficult purification of curcumin and by the presence of two additional yellow components. Food remains the main use for turmeric (old spice); its use as a dye has diminished since the 19th century, but it may have pharmaceutical uses (new spice).